THERAPY

Abstract: The present invention relates to improvements in targetted enzyme prodrug therapy including antibody-directed enzyme prodrug therapy (ADEPT), it particularly relates to novel enzymes and prodrugs for use in ADEPT.

Description: THERAPY The present invention relates to improvements in targetted enzyme prodrug therapy including antibody-directed enzyme prodrug therapy (ADEPT), it particularly relates to novel enzymes and prodrugs for use in ADEPT. In the therapy of certain conditions it is preferable that a drug be delivered only to a particular cellular subpopulation. For example the use of drugs in the treatment of cancer is limited by the inability of the cytotoxic drug to differentiate between cells exhibiting normal cell division and those exhibiting neoplastic division. Hence the therapy is not targeted to a clinically acceptable extent and healthy cells are exposed to cytotoxin. Conjugation of a drug to an antibody, preferably a monoclonal antibody (mAb), allows the targeting of the drug to a particular cellular subpopulation expressing the antigenic determinant to which the targetting antibody binds. However factors, such as the inability of the conjugate to penetrate the relevant tissue, poor release of the drug from the antigen bound conjugate and the limitation placed on the amount of drug which can be delivered by the number of available antibody-binding sites, have limited the effectiveness of this approach. Avoidance of such limitations led to the concept of targeting conjugates of antibodies and enzymes capable of converting relatively non-cytotoxic 'prodrugs* into low molecular weight cytotoxins at the antibody-binding site. This general concept was disclosed by Rose in European Patent application 84302218.7. Bagshawe and collaborators have referred to the concept as ADEPT, Antibody Directed Enzyme Prodrug Therapy. (Bagshawe K.D. et.al., Br. J. Cancer [1987] 56, 531-532, Bagshawe K.D. et al, Br. J. Cancer [1988] 50, 700-703 and WO 90/10140). In this way one conjugate could generate a proportionately larger amount of drug at the target site by repeated rounds of enzymatic catalysis of prodrug activation. EP 382411 describes ADEPT wherein a prodrug may be converted to a cytotoxic agent by enzymes including beta-lactamases isolated from various micro-organisms, L- pyroglutamate aminopeptidase, beta-galactosidase, D-amino acid peptidase, isoenzymes of alkaline phosphatase and various carboxypeptidases. WO 91/11201 describes ADEPT wherein a prodrug may be enzymatically cleaved to generate cyanide by β-glycosidases or β-glucosidases, generally of plant origin. EP 302 473 describes ADEPT wherein the enzyme alkaline phosphatase may be used to cleave novel prodrugs of mitomycin, penicillin N amidase may be used to cleave novel prodrugs of adriamycin, or cytosine deaminase may be used with the prodrug 5-fluorocytosine. EP 484 870 describes ADEPT wherein β-lactamase may be used to activate a cephalosporin prodrug to yield a cytotoxic nitrogen mustard. WO 88/07378 describes ADEPT wherein benzoic acid nitrogen mustard glutamides may be converted to the nitrogen mustard under the action of carboxypeptidases. Nitols, K. S., et al, Pteridines 3, [1992], 125-126, discloses a number of MTX-amino acid prodrugs which may be activated by carboxypeptidase-mAb conjugates as part of ADEPT. WO 91/09134 discloses bispecific hybrid mAbs for use in ADEPT wherein the mAb has specificities against both human cancer cell antigens and a prodrug-activating enzyme. All previously disclosed methods of ADEPT may be divided into two categories: those which employ human enzymes and those which employ non-human (eg. bacterial) enzymes to activate the relevant prodrug. Both strategies retain inherent problems which limit their potential to provide effective therapy. Use of a human enzyme results in instability of the associated prodrug in vivo, as it may be activated at sites distant to the target site where endogenous human enzymatic activity may occur naturally. Clearly this will also have the highly undesirable effect of generating potentially cytotoxic compounds in non-targetted areas of the body with possibly lethal consequences. The use of a non-human enzyme permits the associated prodrug, which is only activated by the non-human enzymatic activity, to avoid activation by endogenous enzymes and so remain stable in vivo until converted to drug at the target site. However, such a non- human enzyme may elicit an immune response when introduced in vivo and antibodies generated to the enzyme will limit or destroy its ability to activate the prodrug. WO 90/07929 identifies the desideratum of a non-endogenous catalytic activity being provided by a non-immunogenic enzyme but teaches only that this may be achieved by the use of "genetically conserved" enzymes or those from a "genetically similar species". However it is not taught how this may be accomplished. It has now been found that the apparent conflict arising from the need to provide a prodrug which is stable in vivo and yet activated by a non-immunogenic enzyme may be resolved by generation of a mutant mammalian enzyme which retains catalytic activity but possesses a novel substrate specificity. The associated prodrug may be activated by the catalytic activity of the mutant enzyme but since the substrate specificity of the mutant enzyme is not a naturally occurring one, the prodrug remains stable in vivo until converted to drug at the target site. The ability to use a non-immunogenic enzyme according to the present invention provides the further advantage that repeated rounds of therapy may be administered. This is in contrast to known processes for ADEPT, in which the initial introduction of the enzyme to the system elicits an immune response which effectively precludes further treatment with the same enzyme as this will be removed from the body by an immune reaction 'primed' during the first round of therapy. One aspect of the present invention provides a method of targetting a chemotherapeutic agent to a specific cell-type comprising the administration to a mammal of (i) an effective amount of a conjugate of a cell-type specific targetting molecule with a mutant mammalian enzyme capable of catalysing a functionally inactive precursor of a chemotherapeutic agent to its active form and (ii) an effective amount of the functionally inactive precursor of the chemotherapeutic agent which is refractory to endogenous catalysis to the chemotherapeutic agent. Endogenous catalysis implies conversion of functionally inactive precursor of a chemotherapeutic agent to that chemotherapeutic agent by enzymes naturally present in vivo. Clearly, conversion occurring at the target site, catalysed by the targetted mutant mammalian enzyme, is not considered to be endogenous catalysis. A further aspect of the present invention provides a method of treatment of a mammal requiring therapy for any of the conditions hereinafter described comprising the administration to the mammal of an effective amount of a cell-type specific targetting molecule conjugated with a mutant mammalian enzyme which is capable of catalysing a functionally inactive precursor of a drug to its active form in combination with an effective amount of a functionally inactive precursor of the drug which is refractory to endogenous catalysis. A mutant human enzyme as used herein shall be taken to be any human enzyme with a sequence differing by at least one amino acid from the amino acid sequence or sequences of that enzyme in the patient to which the therapy is applied. As used herein the term "cell-type" means any localised or dispersed population of cells possessing a common determinant essentially selective for a pathological state, for example, cancer cells expressing carcinoembryonic antigen, Tag-72, mucin, or the antigens recognised by the antibodies Ing-1, 17-1A, 323/A3, R-LU-10, cl74, PR.1A3, MOV18, G250, U36, E48, NR-CO-02 or any of the other antigens shown in Table 3. The cell-type specific targetting molecule may be any molecule capable of being noncovalently or covalently linked or conjugated with the mutant enzyme and which can demonstrate a selectivity in its binding affinity for cell-surface markers. Such molecules include polyclonal and monoclonal antibodies, including bispecific antibodies in which one antibody arm noncovalently binds the mutant enzyme and the other performs the cell-type specific targetting (U. Sahin, et al, Cancer Research 50, 6944, 1990). Alternative targetting molecules which may be linked to mutant mammalian enzymes to form conjugates include hormones, ligands, cytokines, antigens, oligonucleotides and peptidomimetics. The choice of targetting molecule will depend upon the nature of the cells to be targetted but will most probably be an antibody and preferably a monoclonal antibody as mAbs produce the greatest selectivity. A "chemotherapeutic agent" which may also be referred to herein as a "drug" includes any molecule which has activity in human therapy. Such chemotherapeutic agents include but are not limited to cytostatic or cytotoxic compounds used in the therapy of cancers or viral infections. A "functionally inactive precursor" which may also be referred to herein as a "prodrug" includes any compound which may be converted into a chemotherapeutic agent under the action of an enzyme. Such functionally inactive precursors may typically be converted to a chemotherapeutic agent by the enzymatic cleavage of the functionally inactive precursor to yield a chemotherapeutic agent and a "prodrug moiety". Such conversion from functionally inactive precursor to chemotherapeutic agent may also occur by enzymatically mediated isomerisation. A functionally inactive precursor will generally not exhibit clinically significant levels of the therapeutic activity possessed by the chemotherapeutic agent into which it may be enzymatically converted having been chemically derivitized to decrease its normal pharmacological activity. The functionally inactive precursor of a cytotoxic chemotherapeutic agent will not itself exhibit clinically significant cytotoxicity and will be sufficiently stable in vivo such that during therapy, clinically significant level of cytotoxicity are largely only generated at the site of conversion of functionally inactive precursor to chemotherapeutic agent y at the site to which the mutant enzyme has been targetted. The mutant mammalian enzyme is preferably a wild-type mammalian enzyme with one or more mutations which generate novel substrate specificities without engendering a significant immunological response when administered during human therapy. A significant immunological response may be regarded as one which would preclude the clinical use of such an enzyme in human therapy. The essential characteristic of a mutant mammalian enzyme of the present invention is the presence of a mutant substrate-binding site irrespective of the amino acid sequences flanking this mutant substrate-binding site. Hence a chimaeric enzyme comprising a mutant substrate-binding site and flanking sequences derived from one or more different enzymes is included with the definitions of a mutant mammalian enzyme of the present invention. Such a chimaeric enzyme may be generated by recombinant DNA technology and/or protein engineering. Enzymes suitable for directed mutagenesis include any enzymes possessing a catalytic activity capable of converting a prodrug to a drug. Such catalytic activities include transferase, hydrolase, oxido-reductase, isomerase, lyase, or ligase. The directed mutagenesis will generate a novel substrate specificity but preserve the class of catalytic activity involved. For example a mutant isomerase of the present invention will possess a novel isomerase activity sufficiently different from the isomerase from which it was derived to ensure that prodrugs susceptible to activation by the mutant isomerase remain substantially stable in the presence of the isomerase from which the mutant enzyme was derived. As will be shown this dramatic shift in activity may be achieved by the alteration of as little as one residue at the catalytic site. The alteration of the minimum number of residues necessary to obtain the required shift in activity ensures continuity of enzyme structure and hence avoids negative immunological effects when the mutant enzyme is introduced to the body. A preferred mutant enzyme of the present invention is mutant mammalian carboxypeptidase A (CPA). This enzyme has the general activity of cleaving carboxy- terminal aromatic and aliphatic amino acid residues and has been characterised in a diversity of species and tissue-specific variants. Particularly preferred mutant carboxypeptidases include mutants derived from human pancreatic carboxypeptidase Al (Catasus L. et al, Biochem. J. 287. 299-303, 1992), human mast cell carboxypeptidase A (Reynolds D.S. et al. J. Clin. Invest. 89 273-282, 1992) and human pancreatic carboxypeptidase A2 (Fig 9 hereinafter). It will be appreciated that the common characteristic of these carboxypeptidases is the presence of a CPA-like substrate-binding pocket and associated enzymatic activity irrespective of overall sequence or structure of the enzyme and that any enzyme possessing this CPA-like substrate-binding pocket is amenable to mutation to a mutant carboxypeptidase of the present invention. In a particularly preferred embodiment of the present invention the mutant enzyme is mutant human pancreatic carboxypeptidase Al or A2 (CPA1 or CPA2) and yet more preferably, CPA1 or CPA2 wherein amino acid substitutions are generated at one or more of residues 203, 210, 242, 244, 250, 253, 255, 267, 268, 269 and 305, of the amino acid sequences shown in Table 4 which represent the wild-type (w.t.) sequences of CPA1 and CPA2 respectively. Particularly preferred combinations of residue substitution include Gly at residues 250 and 268 when 253 and 255 are w.t.; Gly at 253 and 268 when 250 and 255 are w.t.; Gly at 250 and His at 268 when 253 and 255 are w.t.; Gly at 250 when 253, 255 and 268 are w.t.; Ala at 255 and His at 268 when 250 and 253 are w.t. and His at 268 when 250, 253 and 255 are w.t. The most preferred mutants are carboxypeptidase Al or A2 mutants comprising a single substitution; Gly at The most preferred conjugate of the present invention comprises all or part of either of mAbs CAMPATH-IH®, 323/A3 or ING-1 and at least the substrate-binding domain of either of mutant mammalian enzymes CPA1 or CPA2 having glycine present at amino acid position 268 as described in Table 5. Human pancreatic carboxypeptidase A is expressed as a preproenzyme (Fig. 2) which is processed to a proenzyme and subsequently to the mature enzyme. Mutant carboxypeptidases of the present invention may be mutants of the preproenzyme, the proenzyme or of the mature enzyme but are preferably mutants of the mature enzyme. These mutants may be derived from either preproenzyme, the proenzyme or the mature enzyme but are preferably derived from the proenzyme. The mutated proenzyme (or preproenzyme) may then be converted to the corresponding mutant mature enzyme by standard methods such as trypsinisation. A mutant enzyme of the present invention may be generated from the DNA or RNA source of any enzyme possessing the previously discussed activities by methods well known in the art of molecular biology and more particularly by the methods described hereinafter in the Examples. The selection of the enzymatic activity and the primary sequence of a mutant enzyme of the present invention will clearly depend upon the nature of the relevant prodrug and, if a prodrug of the type which is activated by the removal of a prodrug moiety, on the precise structure of that moiety. The substrate-binding or active site of the mutant enzyme must, unlike the corresponding non-mutant enzyme, be capable of interacting with the prodrug in such a way that enzymatic catalysis is facilitated. The ability of the mutant enzyme to perform this activity will depend upon subtle alterations in the 3-dimensional structure of the active site, which is in turn dependent upon the primary amino acid sequence of this region of the protein. Alterations of the primary sequence of an enzyme by standard techniques of protein engineering will allow the generation of an appropriate mutant enzyme for use according to the invention with a corresponding prodrug. Novel prodrugs comprise those of formula (II) P B-D-E-F-X - C — CO 2 H (II) I R — C — R 2 I R 2a wherein, X represents NH or O and R 2 represents CO2H, SO3H, SO2H, OSO3H, PO3H2, OPO 3 H 2 , C g alkyl esterified CO2H, Cj.6 esterified PO3H2, 04.12 alkyl, C4.12 alkenyl, C4.12 alkynyl, C4.12 branched alkyl, Cj.g alkyl or C3.8 branched alkyl; substituted with CO2H, SO3H, SO H, OSO3H, PO3H2, OPO3H2, C1.6 alkyl esterified CO2H, Cι_ 6 alkyl esterified PO3H2, hydroxyl, alkoxy, halo, haloalkyl, cyano or carboxamide; C3_ cycloalkyl, aryl or heteroaryl optionally substituted with Cj.g alkyl, C3.8 branched alkyl, C3_g cycloalkyl, C3.6 alkenyl, C2-6 alkynyl, C4.6 cycloalkenyl, trisubstituted-silyl i.e. CR 13 )(R 14 )(R 15 )Si, where R 13 , R 14 , and R 15 are C _ 6 alkyl, C 3 . 6 branched alkyl, C 3 _ 6 cycloalkyl, C3.6 alkenyl, C2-6 alkynyl, C4.6 cycloalkenyl, aryl, or heteroaryl, and where R 3 , R* 4 , and ~R\- are each the same group or different groups (e.g. trimethylsilyl, tert-butyldimethylsilyl, cyclohexyldimethylsilyl, or phenyldimethylsilyl), aryl, CO 2 H, SO3H, SO 2 H, OSO3H, PO 3 H 2 , OPO 3 H 2 , Cι_ 6 alkyl esterified CO 2 H, Cj_6 alkyl esterified PO3H2, carboxamide, hydroxyl, Cj_6 alkoxy, C\.β alkythio, mercapto, halo, haloalkyl, nitro or cyano, R a and R D represent H, hydroxy, mercapto, alkoxy, alkylthio, halo, cyano, CO2H, SO3H, SO H, OSO3H, PO3H2, OPO 3 H 2 , Cι_ 6 alkyl esterified CO2H, carboxamide, C\. alkyl esterified PO3H2, cyclic C2.6 [i*e*, R 2a and R 2b together represent (CH2)2-6l » trisubstituted-silyl i.e. (R 13 )(R 14 )(R 15 )Si, where R 13 , R 14 , and R 15 are Cj_6 alkyl, C3.6 branched alkyl, C3.6 cycloalkyl C3.6 alkenyl, C2-6 alkynyl, C4.6 cycloalkenyl, aryl, or heteroaryl and where R* 3 , R^ 4 , and R^ could be the same group or different groups, e.g. trimethylsilyl, tert-butyldimethylsilyl, cyclohexyldimethylsilyl, or phenyldimethylsilyl; or C g alkyl or C\. branched alkyl or C j _6 cycloalkyl or aryl or heteroaryl optionally substituted with hydroxy, mercapto, C\_ 6 alkoxy, C\.β alkylthio, halo, cyano, CO2H, SO3H, SO2H, OSO3H, nitro, PO3H2, OPO3H2, Cj_6 alkyl esterified CO2H, carboxamide, Cι_g alkyl esterified PO3H2, \. alkyl, Cι_6 branched alkyl, C g cycloalkyl, trisubstituted-silyl i.e.(R 13 )(R 14 )(R 15 )Si, where R 13 , R 14 , and R 1 * 5 are C\. alkyl, C3.6 branched alkyl, C3..6 cycloalkyl, aryl, or heteroaryl, and where R^ 3 , R^ 4 , and R^ could be the same group or different groups, e.g. trimethylsilyl, tertbutyldimethylsilyl or phenyldimethylsilyl; with the proviso that R 2a and R 2,3 are not both hydroxy or mercapto. F comprises a moiety of formula (IN) wherein R* represents a group corresponding to the side chain of any α- amino acid for example, H, C\. alkyl, Cι_g alkenyl, Cι_6 alkynyl optionally substituted with CO2H, Cι_6 alkyl esterified CO2H, OPO3, PO3H2, Cι_6 alkyl esterified PO3H2, halo, hydroxy, carboxamide, amino optionally substituted with C\_ alkyl, cyano or nitro, E represents • where n = 3-6 or each of which may be optionally substituted with one or more of hydroxy, one or more of alkoxyl, halo, or C\.(- alkyl optionally substituted with one or more of hydroxy, Cι_6 alkoxy or halo. D represents -CH 2 -CH(R 3 )-, -CH 2 -NR 3 -, -NR 3 -CH 2 -, -CH 2 S- or -CH 2 O- where R 3 is H, Cι_6alkyl, allyl or propargyl, optionally substituted with one or more of C\. alkoxy halo, hydroxy or cyano; and B represents wherein, R 4 represents H, C3.6 branched alkyl, C3.6 cycloalkyl, or C . alkyl; R 5 represents C]_6 alkyl, C3.6 branched alkyl, C3.6 cycloalkyl, amino (optionally substituted with Cι_g alkyl, Cj.g alkanoyl or benzyl); R^ represents H, Cι_6 alkyl, C3.6 branched alkyl, C3.6 cycloalkyl, amino (optionally substituted with Cj_6 alkyl, C\. alkanoyl or benzyl), Cj_6 alkoxyl or C\. alkylthio; and R^ represents H, Cι.g alkyl, C3.6 branched alkyl, C3.6 cycloalkyl, halo C\. alkyl or halo and salts N-oxides, solvates and physiologically functional derivatives thereof; with the proviso that the following compounds are not included within the definition of novel prodrugs although the use of these compounds in therapy does form one embodiment of the present invention: N-(N-(4-(((2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl)(2-propynyl)amino) benzoyl)-L-glutam- 1 -yl)-L-glutamic acid, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-L- glutamic acid, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-L-aspartic acid, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -y 1 -L- phenylalanine, 4-bis(2-chloroethyl)amino)-phenylalanylphenylalanine and 4-bis(2-chloroethyl)arnino)-phenylalanyl-3,5-dimethyl-4-methoxyphenylalanine In a preferred embodiment of prodrugs according to the present invention, X isNH; R 2 is C3_g cycloalkyl or aryl substituted with C j .g alkyl, C3_ cycloalkyl, C3.9 branched alkyl or trisubstituted-silyl i.e. (R 13 )(R 14 )(R 15 )Si, where R 13 , R 14 , and R 15 are C]_6 alkyl, C_,__. branched alkyl, 03.5 cycloalkyl, aryl, or heteroaryl, and where R 13 , R* 4 , and R^ are each the same group or different groups (e.g. trimethylsilyl, tert- butyldimethylsilyl, cyclohexyldimethylsilyl, or phenyldimethylsilyl); R 2a and R 2b are both H; R 1 is H, C!_ 6 alkyl, CH 2 CO 2 H, CH 2 CH 2 CO 2 H or CH 2 ,CH 2 CH2NH 2 , E is D is -CH 2 NH-, -CH 2 N(CH 3 )-, -CH 2 N(CH 2 C≡CH)-, -CH 2 CH 2 - or -CH 2 -CH(C 2 H 5 )- and B is and salts, N-oxides, solvates and physiologically functional derivatives thereof In the most preferred embodiment of prodrugs according to the present invention, X is NH; R 2 is phenyl or para-hydroxyphenyl substituted with cyclopentyl, cyclobutyl or tert- butyl. The cyclopentyl, cyclobutyl and tert-butyl groups may both be either ortho- or meta- but are preferably meta-, R a and R 2b are H, R 1 is CH 2 CH 2 CO 2 H D is -CH,N (CH.) . and B is and salts, N-oxides, solvates and physiologically functional derivatives thereof. Where chiral centres of prodrugs as defined above are present they may, independently, each be either in the S or R configuration or may be a mixture of the S and R configuration. Preferably they are in the S-configuration for T' and the chiral carbon of formula (II) adjacent to X. A preferable prodrug for use according to the present invention is a prodrug of the cytotoxic agent melphalan (UK 750, 155) which is commercially available under the name Alkeran (T. M. The Wellcome Foundation Limited) and has the chemical name 4-[bis(2-chloroethyl)amino]-l-phenylalanine. Prodrugs of melphalan according to the present invention have the general formula wherein R 2 , R 2a , R 2b and X are as herein defined. The following are particularly preferred novel prodrugs according to the present invention: N-((S)-4-carboxy-2-(5-(((l,2-dihydro-3-methyl-l-oxobenzo(F)quinazolin-9- yl)methyl)amino)- 1 -oxo-2-isoindolinyl)butanoyl)-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-2- cyclopentyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl- 3 -cyclopentyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl- 3-cyclobutyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl- 3-trimethylsilyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3 - tert-butyl-L-phenylalanine, N-(4-((3-(2,4-di__π_ino-l,6-dihydro-6-oxo-5-pyrimidinyl)propyl)amino)benzoyl)- L-glutam- 1 -yl-3-cyclopentyl-L-phenylalanine, N-(4-(((l,2-dihydro-3-methyl-l-oxobenzo(F)quinazolin-9-yl)methyl)amino)-2- fluorobenzoyl)-L-glutam- 1 -yl-3-cyclopentyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl-methyl)methylamino)benzoyl)-L-glutam-l-yl-3- cyclopentyl-L-tyrosine, N-(4-(((2,4-di__mino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl- 3 , 5-dϋodo-L-tyrosine, N-(4-(((2,4-di__mino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-2- cyclopentyl-L-tyrosine, (S)-3-(3-cyclopentyl-4-hydroxyphenyl)-2-((N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl)oxy)propionic acid, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3- tert-butyl-L-tyrosine and salts, N-oxides, solvates and physiologically functional derivatives thereof. The most preferred prodrug according to the present invention is: N-(4-(((2,4-di amino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3 - cyclopentyl-L-tyrosine. Any chiral centers of any of the chemical structures on compounds described herein can be in the R and S configuration. Although these chiral centers of the chemical structures drawn hereinafter which are depicted in the preferred S-configuration, it is understood that the corresponding chemical structures having the chiral centers in the R- configuration as well as enantiomeric and diastereomenc mixtures of these compounds are also considered as being party of the invention. A prodrug of formula (V), where B, D and E are as defined in formula (III), and X, R! and R 2 are as defined in formulas (IN) and (II), can be prepared by hydrolysis of an ester of formula (VI); where R^ is alkyl, branched alkyl, or cycloalkyl group such as methyl, ethyl, tert-butyl or cyclohexyl; or an aryl group such as phenyl, 2,6-dimethylphenyl; benzyl (optionally substituted with halo, alkoxy, or alkyl groups such as methyl or tert-butyl); or 9-fluorenylmethyl (Fm); normally in the presence of a base, such as NaOH, LiOH; or, in the case where R -* is Fm, diethylamine or piperidine; or in the presence of an acid such as HCl or trifluoroacetic acid; in a solvent or solvent mixture such as THF, nitromethane or THF:H2θ; at a temperature, for example 0°C to reflux temperature, conveniently at room temperature; or in the present of an acid, such as HCl or trifluoroacetic acid; in a solvent such as THF or nitromethane; at a temperature, for example 0°C to reflux temperature, conveniently at room temperature; or by hydrogenolysis where R*- is 9- fluorenylmethyl (Fm) or benzyl optionally substituted with alkyl groups such as methyl or tert-butyl using hydrogen at ambient or elevated pressure, for example between 1* 75kgcm ~2 and 7*0kgcm" 2 , conveniently at 3*5kgcm" 2 , in the presence of a catalyst such as palladium-on-carbon, in a polar solvent, for example methanol or ethanol, or a mixture of methanol and dichloromethane, at ambient temperature, or by methods which are known to anyone skilled in the art such as those methods described in Rylander, P. Ν., Catalytic Hvdrogenation in Organic Syntheses. Academic Press: New York, 1979, or in Rylander, P.N., Hvdrogenation Methods. Academic Press: London, 1985, or in Bodanszky, M., et.a , The Practice of Peptide Synthesis. Springer-Nerlag: Berlin, 1984, which are incorporated herein by reference; or by oxidation where R^ is phenyl or benzyl substituted with alkoxy (preferably in the 2-, 4-, or 6- position of the phenyl ring) optionally substituted with suitable alkyl groups such as methyl or tert-butyl, in the presence of a suitable oxidizing agent such as eerie ammonium nitrate or dichlorodicyanoquinone (DDQ) in a suitable solvent such as methanol, water, dichloromethane, or combinations thereof, by methods which are known to anyone skilled in the art such as those methods described in Heathcock, OH., et.a , Tetrahedron. 1981, 37, 4087; or those methods described in Greene, T.W., et.al, Protective Groups in Organic Svnthesis. second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference; or where R 8 is 2-(trimethylsilyl)ethyl by reaction with a source of a suitable nucleophile such as potassium fluoride or tetrabutylammonium fluoride for which methods are known to anyone skilled in the art such as those methods described in Greene, T. W., et al, Protective Groups in Organic Synthesis, second edition. John Wiley & Sons: New York, 1991, or in Bodanszky, M., et.al, the Practice of Peptide Synthesis. Springer-Verlag: Berlin, 1984, or in Jones, J., The Chemical Synthesis of Peptides. Clarendon Press: Oxford, England, 1991, which are incorporated herein by reference; or where R 8 is a photoremovable group such as ortho- nitrophenyl by photolytic methods which are known to anyone skilled in the art such as those described in Cama, L.D., et.al., J. Am. Chem. Soc. 1978, 100. 8006, or in Pillai, N.N.R., Synthesis. 1980, 1, or in Pillai, V.N.R., Ore. Photochem.. 1987, 9, 225 which are incorporated herein by reference. Esters present on sidechains R 1 or R 2 may or may not be hydrolyzed in the same reaction mixture utilised to hydrolyze R 8 , depending on the choice of ester group and the reaction conditions. Ester (VI) can be prepared by reaction of an amine of formula (Nil) where Rl is defined as in formula (IN), R 2 and X are defined as in formula (II), and R 8 is defined as in formula (NI); a carboxylic acid of formula (Nπi), B-D-G-CO 2 H (Nπi) where B and D are as defined in hereinbefore and G represents a group (IX), - (CH 2 ) n - (IX) where n = 3-7, or 1,4-cyclohexyl (X) where the 1- and 4-substituents may be either in the cis- or trans- configuration or mixtures of the two configurations, or an aromatic group chosen from the following: The reaction of amines of formula (Nil) with carboxylic acids of formula (Nπi) are carried out in the presence of a coupling agent; including but not limited to diethyl cyanophosphonate (XI), l-ethyl-3-(dimethylaminopropyl)carbodiimide (XTI), or dicyclohexylcarbodiimide (Xlla), optionally in the presence of an activating group such as 1-hydroxybenzotriazole (HOBT), in a polar, aprotic solvent, conveniently Ν,Ν- dimethylformamide (DMF) or N,N-dimethylpropyleneurea (DMPU) at a temperature, for example 0°C to 80°C, II (XI) NCP(OEt) 2 Me 2 N (CH 2 ) 3 N= C*--=N Et pαi) conveniently at room temperature. Alternatively, an ester of formula (VI) can be prepared by treating a mixture of carboxylic acid (Nπi) and a tertiary amine base; such as triethylamine, diisopropylethylamine, or Ν-methylmorpholine; in an aprotic solvent, such as DMF or DMPU; at a temperature from 0°C to 25°C ; with a acylating agent, such as isobutyl chloroformate or ethyl chloroformate; followed by addition of amine (vπ). Several of the preferred carboxylic acids (NTJI) can be purchased from commercial suppliers, eg. (XIII) can be purchased from Aldrich Chemical Co; or can be prepared via literature methods, eg. (XIN): De Graw, J I, et al, J. Med Chem- 1982, 25, 1227; Ibid, 1986, 29, 1056. (XV): Jones, T R, et al,: J. Med Chem- 1986, 29, 1114; Νair, M G, et al-: Jbid, 1986, 29, 1754; Ghazola, M, et al,: Ibid, 1986, 29, 1263; Acharya S.P. et.al, J. Heterocvclic Chem. 1975, U, 1283. (XNI): Bamett, C J, et al, Tetrahedron Lett. 1989, 30, 6291. (XVII): Styles, N L, et al x : J. Med Chem- 1990, 33, 561 Taylor, Ex., et al. J. Med.Chem. 1992, 32 1517: (XNI la) and Bisset, G.M.F. et al J. Med. Chem. 1992 35 859; Carboxylic acids of formula (Nm) can be prepared by hydrolysis of esters of formula (XNIH), wherein B and D are as defined in formula (III), G is as defined in formula (Nm) and R9 is a suitable group such as methyl, ethyl, or tert-butyl; normally in the presence of a suitable base, such as ΝaOH or LiOH; or a suitable acid, such as HCl or trifluoroacetic acid; in a suitable solvent or solvent mixture, such as THF, nitromethane, or THF:H O; at a temperature, for example from 0°C to reflux temperature, conveniently at room temperature. B-D-G-CO 2 -R 9 (XNIII) For prodrugs where D in formula (III) or formula (XNm) represents -CH 2 -ΝR 3 -, esters of formula (XNm) can be prepared by: (a) reaction of a compound of formula (XIX), where G is as defined in formula (Nπi), R 9 is as defined in formula (XNm), and R 10 represents R 10 -G-CO 2 R 9 (XIX) ΝHR 3 wherein R 3 is as defined in formula (III); and a compound of formula (XX) K-R 11 (XX) wherein R^ is an aldehyde group or cyano group and K is a heterocyclic group such as those defined as B in formula (111) or a group such as (XXI) where Z=O, or a group such as defined as B in formula (III) or a group such as (XXI) where Z=O substituted with a suitable protecting group to provide a more suitable reactant, suitable groups including but not limited to Ν-pivaloyl, Ν-benzoyl, Ν- carbobenzyloxy (Ν-cbz), or Ν-tert-butoxycarbonyl (Ν-t-BOC). Such protecting groups can be removed at a later stage in the synthesis, where appropriate, using standard methods known to anyone skilled in the art such as those methods described in Green, T.W., et.el, Protective Groups in Organic Svnthesis. second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference. Esters of formula (XNm) are prepared by reaction of (XNI) and (XX) in a solvent such as THF, DMPU, DMF, ethanol, or acetic acid at a temperature between 25°C and 100°C, optionally with removal of water during the course of the reaction, followed by reduction with sodium cyanoborohydride or hydrogen gas in the presence of a suitable catalyst, such as Raney nickel, using standard reaction conditions known to anyone skilled in the art. (b) reaction of a compound of formula (XIX), as defined in (a), with a compound of formula (XX), where K is as defined in (a) but R^ 1 is defined as CH 2 Y, where Y is defined as a leaving group such as chloro, bromo, iodo, mesyl, or tosyl, in a suitable solvent such as THF, in the presence of a base such as ΝaHCθ3, Na 2 CO3, K 2 CO3, triethylamine, or diisopropylethylamine, at an elevated temperature, for example 50°C to 150°C and conveniently 80°C to 120°C. For prodrugs where D in formula (HI) and formula (XNm) represents -ΝR 3 -CH 2 -, esters of formula (XVm) can be prepared by reaction of a compound of formula (XIX) where R^ represents an aldehyde group and G is as defined in formula (NIII), and a compound of formula (XX), where K is as defined in formula (XX) and R* ** is defined as HΝR 3 where R 3 is as defined in formula (III), in a solvent such as THF, DMF, DMPU, or acetic acid at a temperature between 25°C and 100°C, optionally with removal of water during the course of the reaction, followed by reduction with sodium cyanoborohydride or hydrogen gas in the presence of a suitable catalyst, such as Raney nickel. For prodrugs where D in formula (HI) and formula (XNm) represents -CH 2 O-or-CH 2 S- esters of formula (XNm) are prepared by reaction of a compound of formula (XIX), where R 9 is as defined in formula (XNm), G is as defined in formula (Nm), and R 10 is OH or SH; with a compound of formula (XX), where K is as defined in formula (XX) and R! is CH2Y, where Y is defined as a leaving group such as chloro, bromo, iodo, mesyl, or tosyl; in a suitable solvent such as DMF, DMPU, or acetone; in the presence of a base such as triethylamine, diisopropylethylamine, cesium carbonate, cesium bicarbonate, sodium carbonate, or sodium bicarbonate, at a temperature from 25°C to 80°C. For prodrugs where D in formula (III) and formula (XvTfl) represents -CH2CH2-, esters of formula (XNm) are prepared by: (a) reaction of a compound of formula (XX) where K is defined as in formula (XX) and where R* * • is CH2Br, with triphenylphosphine in a polar solvent, for example a C]_4 alkanol or glycol, conveniently methanol or ethanol, normally in the presence of a base, for example a metal alkoxide conveniently formed from the metal and the solvent, ie. sodium methoxide or sodium ethoxide at a temperature of 0°C to reflux temperature; followed by addition of a compound of formula (XIX), where R9 is as defined in formula (XNm), G is as defined in formula (Nm), and RlO is an aldehyde group; followed by reduction with hydrogen at elevated pressure, for example between l-75kgcm ~2 and 7-0kgcnr 2 , conveniently at 3-5kgcm" 2 in the presence of a suitable catalyst such as palladium-on-carbon, in a polar solvent, for example methanol or ethanol or a mixture of methanol and dichloromethane, at ambient temperature. (b) reaction of a compound of formula (XIX) where R9 is defined as in formula (XNm) and G is defined as in formula (Nlϋ), and R 10 is CH 2 Br, with triphenylphosphine in a polar solvent, for example a Cι_4 alkanol or glycol, conveniently methanol or ethanol, normally in the presence of a base, for example a metal alkoxide conveniently formed from the metal and the solvent, ie. sodium methoxide or sodium ethoxide at a temperature of 0°C to reflux temperature; followed by addition of a compound of formula (XX), where K is as defined in formula (XX), and R^ is an aldehyde group; followed by reduction with hydrogen at elevated pressure, for example between l-75kgcm" 2 and 7*0kgcm" 2 , conveniently at 3-5kgcm ~2 , in the presence of a suitable catalyst such as palladium-on-carbon, in a polar solvent, for example methanol or ethanol or a mixture of methanol and dichloromethane, at ambient temperature. (c) reaction of a compound of formula (XX), where K is defined as in formula (XX) and R! is chloro, bromo, or iodo; with a compound of formula (XIX), where R 9 is defined as in formula (XNm), G is as defined in formula (VIII), and R}® is a acetylene group C≡CH. The reaction is carried out in the presence of a Pd(O) catalyst, conveniently tetrakis- (triphenylphosphine)palladium; and a base such as triethylamine or diisopropylethyl- amine; in a polar aprotic solvent such as DMF or DMPU; at a temperature of 25°C to 50 °C; followed by reduction with hydrogen at elevated pressure, for example between 1.75kgcm" 2 and 7.0kgcm" 2 , conveniently at 3.5kgcm ~2 , in the presence of a suitable catalyst such as palladium-on-carbon, in a polar solvent, for example methanol or ethanol or a mixture of methanol and dichloromethane, at ambient temperature. For prodrugs where D in formula (HI) and formula (XVm) represents -CH2-CH(R 3 )-, R 3 is as defined in formula (D ), esters of formula (XNm) are prepared by reaction of a compound of formula (XX), where K is defined as in formula (XX) and where R * is CH2Br, with tributylphosphine or triphenylphosphine in a polar solvent, for example a Cι_4 alkanol or glycol, conveniently methanol or ethanol, normally in the presence of a base, for example a metal alkoxide conveniently formed from the metal and the solvent, ie. sodium methoxide or sodium ethoxide, or in a solution of dimethyl sulfoxide and a base such as sodium hydride at a temperature from 25°C to 80°C; followed by addition of a compound of formula COX), where R is defined as in formula (XVm), G is defined as in formula (NH ), and R^ is a keto group -(CO)-R 3 where R 3 is defined as in formula (III); subsequently stirring the reaction mixture at a temperature from 25°C to reflux temperature for an extended period from 4 to 96 hours, conveniently 24 to 48 hours; followed by reduction with hydrogen at elevated pressure, for example between 1.75kgcm" 2 and 7.0kgcm" 2 , conveniently at 3.5kgcm -2 , in the presence of a suitable catalyst such as palladium-on-carbon or platinum oxide, in a polar solvent, for example methanol ethanol or acetic acid. Compounds of formula (XX) where K is as defined above and R 1 is CH2Br can be prepared using methodology available to those skilled in the art. (Piper, J R et al, J Org Chem. 1977, 42, 208; Piper, J R et al, J Med Chem. 1986, 29, 1080; Oakes, N, et al, J Chem Soc. 1956, 4433; Acharya, S P, et al, J Heterocvclic Chem. 1975, \2, 1283; Bird, O D , et al in Pteridine Chemistry. , Pfleiderer W & Taylor E C, Eds, Pergamon Press, Oxford, 1964, p. 417: Piper, J R, et al, J Heterocvclic Chem. 1974, 279.) Compounds of formula (XX) where K is as defined above and R** 1 is an aldehyde group can be prepared using methodology available to those skilled in the art. (Taylor, E C, et al, J Org Chem. 1983, 48, 4852; Arnold. A, et al, Collect Czech Chem Commun. 1960, 25, 1318; Beardsley, G P, et al, Proc Am Assoc Cancer Res. 1986, 1027.) Compounds of formula (XX) where K is as defined above and R^ is a cyano group can be prepared using methodology available to those skilled in the art. (Elsager, E F, et al, Lect Heterocvclic Chem. 1974, 2, S-97; Davoll, J, et al, J Chem Soc (O. 1970, 997; Jones, T.R., Eur. J. Cancer. 1981, J_7, 11; Kondo, T., etal, Chem. Lett.. 1980, 559. Compounds of formula (XX) where K is as defined above and R^ is an amino group can be prepared using methodology available to those skilled in the art. (Hynes, J B, et al, J Med Chem. 1975, 18, 632, 1191; Davoll, J, et al, J Med Chem. 1972, 15, 837; Bernetti, R et al, J Ore Chem. 1962, 27, 2863.) Compounds of formula (XX) where K is as defined above and R^ is a bromo, chloro, or iodo group can be prepared using methodology available to those skilled in the art. Taylor, E C, et al, J Med Chem. 1992, 35, 4450; Taylor, E C, et al, J Ore Chem. 1989. 54, 3 618; Jones, J H, et al, J Med Chem. 1968, H, 322 ) Carboxylic acids of formula (Nm) where A is a group of formula (XXI) where Z=S, can be prepared from carboxylic acids of formula (Nm) where C is a group of formula (XXI) wherein Z=O, by reaction with a sulfurating agent such as P2S5 in a solvent such as pyridine at a temperature between 25°C and reflux temperature. Compounds of formula (XTX) where G is defined as in formula (NIII), R 9 is as defined in formula (XNm), and R 10 is ΝHR 3 where R 3 is as defined in formula (III) can be prepared from compounds of formula (XIX) where G and R9 are as defined above and RIO is H2 by reaction with a compound R 3 -Y where Y is defined as a leaving group such as chloro, bromo, iodo, mesyl, or tosyl; in the presence of a suitable base such as 2,6-lutidine, in a polar aprotic solvent such as DMF or dimethylacetamide at an elevated temperature between 50°C and 100°C. Compounds of formula (XIX) where G is a phenyl or furyl group, R 10 is NH , and R 9 is defined as in formula (XNm) can be obtained from commercial suppliers, or can be readily prepared from such compounds that are available from commercial suppliers using methods known to anyone skilled in the art. One such preferred compound of formula (XXII) can be prepared by methods described by N Soundararajan and M S Platz. (Soundararajan N et al, J Org Chem. 1990, 55, 2034.) One such preferred compound is of formula (XXIIa) and can be prepared by methods described in Mackay, D., Can J. Chem. 1966, 44, 2881; or in Paul, H., etal. Arch. Pharm. 1978, 52 538. Compounds of formula (XIX) where G is a cyclohexyl group, R^ is NH 2 , and R 9 is as defined in formula (XvTII) can be obtained from commercial suppliers, or can be readily prepared using standard methods available to anyone skilled in the art. (Banfi, A, et al, Svnth Commun. 1989, \9, 1787; Krieg, R, et al, J Prakt Chem. 1987, 329, 1123; Johnston, T P, et al, J Med Chem. 1977, 20, 279.) Compounds of formula (XIX) where G is a thienyl group, T ® is NH 2 , and R 9 is as defined in formula (XNQI) can be prepared by methodology available to anyone skilled in the art. (Goddard, C J, J Heterocvclic Chem. 1991, 28, 17; Decroix, B, et al, J Chem Res (S). 1978, 134; Paul, H, et al, Arch Pharm. 1978, 31_L, 679; Mackay, D, Can J Chem. 1966, 44, 2881.) Compounds of formula (XTX) where G is a thiazole group, R 10 is ΝH 2 , and R 9 is as defined in formula (XVIII) can be prepared from 5-nitrothiazole-2-carboxylic acid (Strehlke, P, Chem Ber. 1973, 106. 721) using esterification and reduction methods known to anyone skilled in the art. Compounds of formula (XIX) where G is a pyridyl group, R 10 is NH 2 , and R 9 is as defined in formula (XNm) can be prepared using methodology available to anyone skilled in the art. (Dawson, M I, et al, J Med Chem- 1983, 26, 1282; Finch, N, et al, J Med Chem- 1978, 21, 1269; Cooper, G H, et al, J Chem SocCC.. 1971, 3257; Deady, L W, et al, Aust J Chem. 1971, 24, 385.) Compounds of formula (XIX) where G is a pyrimidyl group, R^ is NH 2 , and R 9 is as defined in formula (XNm) can be prepared using methods analogous to those described by P R Marsham, et al. (Marsham, P R, et , J Med Chem. 1991, 34, 1594.) General methods for synthesis of compounds of formula (NUI) are described in two recent reviews. (Palmer, D C, et al, Prog Med Chem. 1988, 25, 85; Rosowsky, A, Prog Med Chem. 1989, 26, 1.) Esters of formula (NI) where E is defined as can be prepared by reaction of a compound of formula (XX) wher R* * is CH2Br; and a compound of formula (XXHI) where R is defined as in formula (IV), R 2 and X are defined as in formula (II), and R 8 is defined as in formula (VI); in a polar solvent, such as DMF or DMPU; in the presence of a base, such as sodium bicarbonate, cesium bicarbonate, sodium carbonate or cesium carbonate; at an elevated temperature, for example from 50°C to 100°C, conveniently at 100°C. Compounds of formula (XXm) can be prepared from compounds of formula (VII) and a compound of formula (XXIV) by methods analogous to those described by W Pendergast, et al (Pendergast, W, et al, J Med Chem. [1994], 37 p 838) or in patent application WO 01/19700 which is incorporated in its entirety by reference herein. Compounds of formula (VII) can be prepared from compounds of formula (XXV), where is as defined in formula (IV), R 2 and X are as defined in formula (II), R 8 is as defined in formula (VI), and R** 2 represents a suitable protecting group such as Ν-tert- butoxycarbonyl(Ν-t-Boc), or N-carbobenzyloxy (N-Cbz), for which methods are known to anyone skilled in the art such as those methods described in Greene, T.W., et.al, Protective Groups in Organis Synthesis, second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference. Compounds of formula (XXV) can be prepared by a reaction of carboxylic acid of formula (XXVI) and a compound of formula (XXVII), where X is defined as in formula (II); for compounds where X is NH, compounds of formula (XXV) can be prepared by reaction of a carboxylic acid of formula (XXVI) and an amine of formula (XXVTI), where X is NH, in the presence of a suitable coupling agent such as l-(3-dimethylaminopropyl)-3-ethylcarbodiimide or dicyclohexylcarbodiimide, in the presence of a suitable base such as N-methylmorpholine, in an aprotic solvent such as dichloromethane or DMF at 0°C to 50°C or by methods which are known to anyone skilled in the art such as those mthods described in Bodanszky, M., et.al, The Practice of Peptide Synthesis. Springer- Verlag: Berlin, 1984, or in Jones, J., The Chemical Synthesis of Peptides. Clarendon Press: Oxford, England, 1991, which are incorporated herein by reference. (XXVII) dichloromethane or DMF at 0°C to 50°C for which methods are known to anyone skilled in the art such as those methods described in Greene, T.W., et.a.1, Protective Groups in Organic Synthesis, second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference. For compounds of formula (XXV) where X is O, compounds of formula (XXV) can be prepared by reaction of a carboxylic acid of formula (XXVI) with a suitable activating agent such as para-toluenesulfonyl chloride or benzenesulfonyl chloride, in a suitable solvent such as pyridine or lutidine, at a temperature between 0°C and 25°C; followed by addition of an alcohol of formula (XXVII) where X is O. Esters of formula (XXVII), where X is defined as in formula (II), can be prepared from carboxylic acids of formula (XXVm) using standard methods known to anyone skilled in the art for which methods are known to anyone skilled in the art such as those methods described in Greene, T.W., et.al, Protective Groups in Organic Synthesis, second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference. Carboxylic acids of formula (XXVm), where X is O, can be made from carboxylic acids of formula (XXVm) where X is NH by methods analogous to those described by C Morin, et al (Morin, C, et al, Svnthesis. 1987, 479) and K Mori, et al (Mori, K, et al, Tetrahedron. 1982, 38, 3705). Carboxylic acids of formula (XXVTJI) where X is NH can be prepared from compounds of formula (XXIX); where W is a leaving group such as chloro, bromo, iodo, mesyl, or tosyl and R 2 is defined as in formula (II); by methods analogous to WCH 2 R 2 (XXIX) described by R M Williams (Williams, R M, Aldrichimica Acta. 1992, 25, 11 and references cited therein) , or by M J OTJonnell, et al, (OTDonnell, M J, et al, J Am Chem Soc. 1989, 111, 2353.) Compounds of formula (XXIX), where W is a leaving group such as chloro, bromo, iodo, mesyl, or tosyl can be prepared from compounds of formula (XXIX) where W is a hydroxyl group for which methods are known to anyone skilled in the art such as those methods described in March, J., Advanced Organic Chemistry, fourth edition. John Wiley & Sons: New York, 1992, which are incorporated herein by reference. Some of the preferred compounds of formula (XXIX) where R 2 is. a phenyl group substituted with Cj.g alkyl, C3_g branched alkyl, or C3_ cycloalkyl can be prepared by reaction of a compound of (XXIX) where W is a hydroxyl group, or a hydroxyl group with a suitable protecting group which can be attached and later removed where appropriate in the synthesis, by standard methods known to anyone skilled in the art, and R 2 is a phenyl group substituted with an iodo group or a bromo group; the reaction can proceed by addition of a suitable olefinic compound in the presence of a Pd catalyst by methods analogous to those described by H A Dieck, et al, (Dieck, H A, et al, J Am Chem Soc. 1974, 96, 1133), followed by reduction with hydrogen gas in the presence of a suitable catalyst using standard conditions known to anyone skilled in the art for which methods are known to anyone skilled in the art such as those methods described in Rylander, P.N., Catalytic Hvdrogenation in Organic Syntheses. Academic Press: New York, 1979, or in Rylander, P.N., Hvdrogenation Methods. Academic Press: London, 1985, which are incorporated herein by reference; or the reaction can proceed by reaction with a suitable aklyllithium reagent such as n-butyllithium reagent, in an ethereal solvent such as THF, at a reduced temperature, for example -100°C to -20°C, conveniently at -78°C, followed by addition of the resulting benzylonic alcohol group by methods analogous to those described by C T West, et al, (West, C T, et al, J Org Chem. 1973, 38, 2 675). Some of the preferred compounds of formula (XXIX) where R 2 is a phenyl group substituted with a carboxyl or esterified carboxyl group can be prepared by reaction of a compound of formula (XXIX) where W is a hydroxyl group and R 2 is a phenyl group substituted with a bromo or iodo group, with carbon monoxide in a suitable alkanol solvent by methods analogous to those described by A Schoenber, et al, (Schoenber, A, et al, J Org Chem. 1974, 39, 3318), followed by hydrolysis of the resulting ester, if desired, for which methods are known to anyone skilled in the art such as those methods described in Greene, T.W., et.al, Protective Groups in Organic Synthesis, second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference. Some of the preferred carboxylic acids of formula (XXVm) where X is NH and R 2 is a para-hydroxyphenyl group substituted with Cj.g alkyl, C3_g branched alkyl, or C3_ cycloalkyl can be prepared by reaction of tyrosine substituted on the phenyl ring with a bromo or an iodo group; where the amino and/or carboxyl groups can be optionally substituted with a suitable protecting group which can be attached and later removed where appropriate in the synthesis, for which methods are known to anyone skilled in the art such as those methods described in Greene, T.W., et.al, Protective Groups in Organic Svnthesis. second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference with a suitable olefinic compound in the presence of a Pd catalyst by methods analogous to those described by H A Dieck, et al, (Dieck, H A, et al, J Am Chem Soc. 1974, 96, 1133), followed by reduction with hydrogen gas in the presence of a suitable catalyst using standard conditions known to anyone skilled in the art. Some of the preferred carboxylic acids of formula (XXVm) where X is NH and R 2 is a para-hydroxyphenyl) group substituted with carboxyl or esterified carboxyl can be prepared by reaction of tyrosine substituted on the phenyl ring with a bromo or an iodo group; where the amino and/or carboxyl groups can be optionally substituted with a suitable protecting group which can be attached and later removed where appropriate in the synthesis, for which methods are known to anyone skilled in the art such as those methods described in Greene, T.W., etal, Protective Groups in Organic Synthesis, second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference; with carbon monoxide in a suitable alkanol solvent by methods analagous to those described by (Schoenberg, A, et al, J Org Chem. 1974, 39, 3318), followed by hydrolysis of the resulting ester, if desired, by using standard methods known to anyone skilled in the art. An alternate method for the synthesis of compounds of formula (VI) involves reaction of a compound of formula (XX), where K is as defined in formula (XX) and R 1 * is an aldehyde group, a cyano group, NHR 3 where R 3 is as defined in formula (III), or a group CH2Y where Y is defined as a leaving group such as chloro, bromo, iodo, mesyl, or tosyl; with a compound of formula (XXX), where R 1 is as defined in formula (IN), R 2 and X are as defined in formula (II), R 8 is as defined in formula (VI), G is as defined in formula (Nm), and R 10 is defined as OH, SH, ΝHR 3 where R 3 is as defined in formula (m), an aldehyde group, a keto group -(CO)-R 3 where R 3 is as defined in formula (IE), or a group CH2Y where Y is a leaving group such as chloro, bromo, iodo, mesyl, or tosyl; by methods analogous to those described for the preparation of compounds of formula (XNm) by reaction of compounds of formula (XX) with compounds of formula (XIX). Compounds of formula (XXX) where R* is as defined in formula (IV), R 2 and X are as defined in formula (II), R 8 is as defined in formula (VI), G is defined as in formula (Vm), and RlO is CH2Y where Y is a leaving group such as chloro, bromo, iodo, mesyl, or tosyl can be prepared from compounds of formula (XXX) where R*, R 2 , R 8 , X and G are as defined above, and where ~ R\- is CH2OH for which methods are known to anyone skilled in the art such as those methods described in March, J., Advanced Organic Chemistry, fourth edition. John Wiley & sons: New York, 1992, which are incorporated herein by reference. Compounds of formula (XXX), where R 1 is as defined in formula (IV), R 2 and X are as defined in formula (II), R 8 is as defined in formula (VI), G is as defined in formula (Vm), and R 10 is defined as OH, SH, CH 2 OH, NHR 3 where R 3 is as defined in formula (m), an aldehyde group, or a keto group -(CO)-R 3 can be prepared from compounds of formula (VII), where R Ϊ , R 2 , R 8 , and X are as defined above, with a compound of formula (XIX), where R 9 is hydrogen and R*° is as defined above, by methods analogous to those described for the preparation of compounds of formula (VI) by reaction of compounds of formula (VII) with compounds of formula (VIII). In cases where R 10 is OH, SH, CH2OH, NHR 3 , it may be preferable to add a protecting group to RlO prior to reaction with a compound of formula (VII). Such protecting groups can be added and can be removed at a later stage in the synthesis, for which methods are known to anyone skilled in the art such as those methods described in Greene, T.W., et.al, Protective Groups in Organic Svnthesis. second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference. Compounds of formula (XIX), where R 9 is hydrogen and R^ is defined as OH, SH, CH2OH, NHR 3 where R 3 is as defined in formula (D ), an aldehyde group, or a keto group -(CO)-R 3 above, can be prepared from compounds of formula (XIX), where R 9 is as defined in formula (XVm) and R^ is as defined above, for which methods are known to anyone skilled in the art such as those methods described in Greene, T.W., et.al, Protective Groups in Organic Synthesis, second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference. Prodrugs of general structure (XXXI) where X and R 2 are as defined in formula (II) can be prepared from compounds of formula (XXXII) wherein R* 2 is as defined in formula (XXV), R 2 is as defined in formula (II), and R 8 is as defined in formula (VI), for which methods are known to anyone skilled in the art such as those methods described in Greene, T.W., et.al, Protective Groups in Organic Svnthesis. second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference. Compounds of formula (XXXII) can be prepared by reaction of compounds of formula (XXXiπ) where R 12 is as defined in formula (XXV); with compounds of formula (XXNII) where X is ΝH or O, R 2 is as defined in formula (II), and R 8 is as defined in formula (VI); using methods analogous to those described for the preparation of compounds of formula (XXV) from compounds of formulas (XXVI) and (XXVII). Compounds of formula (XXXm), where R^ 2 is a suitable protecting group as defined in formula (XXV), can be prepared for which methods are known to anyone skilled in the art such as those methods described in Greene, T.W., etal, Protective Groups in Organic Svnthesis. second edition. John Wiley & Sons: New York, 1991, which are incorporated herein by reference; from melphalan (UK 750, 155) which is commercially available under the name Alkeran (T.M. The Wellcome Foundation Limited) and has the chemical name of 4-[bis(2-chloroethyl)amino]-l-phenylalanine. The term protein engineering has come to mean the structurally directed modification of a protein to obtain a specific, desired new set of protein properties or, in this context, a precise enzymatic activity. Two approaches to modification of protein specificity and reactivity can be used. The differences in these approaches depend upon the chemical and structural information available for a specific enzyme. If no chemical structural information is known about a protein, random mutations can be introduced singly or in groups to determine the importance of specific amino acid residues using techniques well known in the art of molecular biology. However, a more directed approach using site directed mutagenesis, can be performed, if there is detailed chemical and structural information available on a protein. This involves only specific changes to amino acid residues that the chemical and structural data suggest are the residues critical to determination of the desired enzymatic activity. If 3-dimensional structural data are available, the methodology can be carried further to provide molecular models of proposed mutations. These models can provide estimates of the relative stability or reactivity of a specific site directed mutation. Typically, in the computer-assisted protein engineering approach, computer models based upon x-ray crystallographic structures or models built, by homology model building, from x-ray structures of homologous proteins are used. In many cases the starting crystallographic structures can be obtained from the publicly available Brookhaven Protein Database. Extensive studies of protein structure and the function of individual amino acids have allowed the classification of residues in respect of their importance in structure, stability, reactivity or catalysis. Many active site amino acid residues in proteins show unusual chemical reactivity, these chemical reactivities characterise the catalytic properties of proteins. This chemical reactivity aids in the identification of the active site and the non- reactive associated residues in the active site. Crystallographic studies in concert with chemical reactivity information can indicate distinctly the active site locations. Additionally, some proteins will crystallise with substrates or inhibitors in the active site. All of these pieces of structural information can be combined to enhance the focus of design efforts towards specific residues as candidates for site directed mutagenesis. The specific desired change in protein reactivity can be obtained by specific changes in the amino acid at a given site of interest. Amino acid residues can be inserted, thus separating functional groups in space, or existing residues can be deleted, bringing functional groups closer together. Additionally, changes in protein structure can be effected by changing the side chain or main chain torsion angles and the functional groups on the amino acid residues. These torsion angles can be changed by exchange, deletion or insertion of amino acid residues. If structural data are available, all these changes can be modelled using computer-assisted molecular modelling techniques. Forces interacting on the protein structures can be modelled in the computer using molecular mechanics or molecular dynamics force fields. The space available for ligand binding within a protein can be modified by changes in the bulk of amino acid side chains. Mutating bulky residues, such as isoleucine or leucine, to alanine or glycine, can provide pockets within a ligand binding site without appreciably changing the hydrophobicity of the site. The opposite mutation can exclude bulky substrates from binding to a specific protein. Mutations that involve changes in the functional groups of the amino acids that make up an active site can provide more drastic changes than bulk tolerance or steric changes. Examples of many of these types of changes are available in the literature (see, eg: Recktenwald, A, et al, J. Biotech, 1993, 28, 1-23 and references cited therein; and Lesk, A , et al, "Antibody Engineering: A Practical Guide", Ed. C. A K. Borrebaeck et al, 1991, 1-75). In systems with high quality structural data, specific hydrogen bonding groups can be inserted to improve the interaction of polar substrates. Replacing uncharged residues with arginine, lysine, or histidine inserts groups that could interact with negatively charged substrates. Replacing residues with glutamic acid or aspartic acid can provide interactions with positively charged substrates and thus change the specificity of an enzyme substrate interaction. In a further aspect of the present invention there is provided a recombinant conjugate comprising a targetting antibody as hereinbefore described and a mutant enzyme of the present invention; such recombinant conjugate having been expressed from a single DNA construct or translated from a single RNA transcript. Also included within the scope of the present invention are the corresponding DNA and RNA molecules encoding such a conjugate. Preferred embodiments of such a conjugate reflect a combination of the preferred embodiments for its two component activities as hereinbefore expressed. Such recombinant conjugates may be produced by expression of fusion constructs, generated by joining, in vitro, fragments of DNA that separately encode the enzyme and all or a part of a mAb that confers antigen specificity (Pastan, I. et al, (1986) Cell, 47, 641-648). Such fusions may be constructed by standard techniques of recombinant molecular biology in ways that place the substrate-binding or active site of the polypeptide at the amino terminus or the carboxy terminus and preferably at the amino terminus. The mAb portion of the construct may be designed so that a single chain species of the antibody is connected to the enzyme portion either with the variable light or the variable heavy domain at the 5'- end. In either case, the variable light and variable heavy gene regions will be connected with a synthetic DNA fragment encoding a peptide linker that achieves the correct spatial arrangement of the antibody variable regions. Such a synthetic DNA region connecting the antibody portion of the fusion construct to the enzyme portion may also be desirable. In other forms, the gene encoding the enzyme may be fused to the 5'-end of the variable region of the heavy or light chain gene that extends to form a Fab or F(ab')2 species. Other variations of the heavy chain gene may be used to form the enzyme/antibody recombinant conjugate. Such variations may include natural heavy chain isotypes, modified full-length heavy chain genes of all antibody isotypes, and heavy chains with various deletions, specifically, deletion of the Cjj2 domain may confer desirable pharmacokinetic properties. Alternatively, the fusion may produce a covalent bispecific antibody: enzyme conjugate where one arm of the antibody is replaced by the enzyme in a fashion similar to but not limited to that described in De Sutter, K and Fiers, W. Mol Immunol 31 261 (1994). The term 'conjugate' as used herein includes such recombinant conjugates. Antibodies designed to have particular cellular specificities may be generated in any way well known in the art. Fragments of antibodies generated by recombinant DNA technology may also be used as targetting molecules. Although the preferred antibodies for use will be those expressed from natural or recombinant human derived cell-lines, it is also possible to exploit antibodies from other antibody producing mammalian cell-lines providing such antibodies are not immunogenic to an extent liable to interfere significantly with therapy or that the antibodies have been 'humanised' such that they no longer elicit an immune reaction in vivo. Such a humanised antibody may be a chimaeric antibody (Morrison et al P.N.A.S. (1984), 8J_, 6851-6855; Boulianne et al, Nature, 1985, 314, 268-270 and Neuberger et al, Nature, 1985, 314, 268-70), or a CDR-grafted antibody (James et al, Nature, 1986, 321, 522-525; Riechmann etal, Nature, 1988, 332, 323-327). An antibody for use according to the present invention preferably is a monoclonal antibody or a fragment thereof. The antibody may therefore comprise a complete antibody, a (Fab') 2 fragment, a Fab' fragment, a Fab fragment, a light chain dimer or a heavy chain dimer or single chain species comprising the variable regions from heavy and light chains. The antibody may be an IgG such as IgGl, IgG2, IgG3, or IgG4; or IgM, IgA, IgE or IgD. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. The antibody may be a chimaeric antibody of the type described in WO 86/01533. A chimaeric antibody according to WO 86/01533 comprises an antigen binding region and a non-immunoglobulin region. The antigen binding region is an antibody light chain variable domain and or heavy chain variable domain. Typically the chimaeric antibody comprises both light and heavy chain variable domains. The non-immunoglobin region is fused to the C -terminus of the antigen binding region. The non-immunoglobulin region is typically a non-immunoglobulin protein and is preferably an enzyme region. The two regions of the chimaeric antibody may be connected via a cleavable linker sequence. Antibodies of any of the classes referred to above may be raised against any antigen characteristic of and essentially limited to, a particular target. Such targets include all types of human pathogens, cells expressing antigens as a consequence of transformation or viral infection, cells expressing particular histocompatibility antigens, cells involved in inflammatory responses, blood clots which may be targetted by antibodies against fibrin, etc. Preferred antibodies for use according to the invention, the antigens which they target and the associated indication are detailed in fig 8. Particularly preferred antibodies for use according to the invention include CAMPATH 1H®, ING-1, cl74, PR1.A3, 323/A3, G250, MOV18, 17-1A, NR-LU-10, U36, NR-CO-O2 as well as antibodies targetting mucins or carcinoembryonic antigen. For the treatment of humans when the targetting moiety is an antibody, it is preferred to use and antibody which does not carry with it the risk of the development of an immune reaction against the antibody itself. Accordingly whilst it is possible to use mouse or rat mono-clonal antibodies it is preferred to use antibodies which have been produced by recombinant DNA technology and which have been engineered to reduce the risk of causing an immune reaction. Thus it is possible to use a chimaeric antibody in which the constant domains of a mouse or rat antibody have been replaced by the constant domainns of a human antibody. However, it is preferred to use a humanised or CDR- grafted antibody for the treatment of humans, i.e. an antibody in which the complementarity determining regions from a mouse or rat antibody are combined with framework regions and constant domains from one or more human antibodies. In one preferred embodiment the method according to the invention is carried out using the CDR-grafted antibody CAMPATH-IH (see Riechmann et al Nature. 322, 323-327 (1988)). As noted above, the antibody CAMPATH-IH can be produced in rate myeloma cells as originally described or it can be produced in any other expression system, particularly an expression system suitable for the production of a correctly folded, glycosylated, mammalian protein. High yields of CAMPATH-IH have been obtained by expression in a genetically manipulated CHO cell line (Page and Sydenham, Biotechnology- 9, 64-68 (1991)). The conjugation of the targetting molecule to the enzyme may be carried out in any appropriate way which will neither interfere with the binding specificity and capability of the targetting molecule nor inhibit the catalytic activity of the enzyme. In the case of proteinic targetting molecules the conjugation may be carried out either by direct covalent linkage following the generation of reactive groups by treatment with protein modifying agents or by the use of homo or heterobifunctional cross-linking molecules. Commercially available linkers with amino or hydrazide groups may be reacted with aldehydes generated by oxidation of the sugar moieties of glycoproteins and carbohydrates to form a Schiffs base (Sela M. et al, Conjugates of Antibodies with Cytotoxic Drugs. Immunoconjugates. Antibody Conjugates in Radioimaging and Therapy of Cancer (C.-W. Vogel, ed.) 189-216, (1987)). Alternatively linkers may be reacted with amino, carboxyl or sulfhydryl groups of the antibody or enzyme. (Wawrzynczak P.E. et al, Methods for Preparing Immunotoxins: Effect of the Linkage on Activity & Stability In : Immunoconjugates : Antibody Conjugates in Radioimaging and Therapy of Cancer (C. - W. Vogel, ed.) 78-85, (1987)). Covalent linkage may be obtained by the generation of sulfhydryl groups and, particularly in the case of antibodies, by reduction of disulfide bonds. Such free sulfhydryl groups may be reacted with haloalkyl groups, p-mercuribenzoate groups and groups capable of Michael-type addition reactions, such as maleimides or groups as per (Mitra et al, J Amer Chem Soc, 101, 3097-3110, 1979). Another method for linking enzyme to antibody comprises utilising the epsilon amino groups of lysine. Reactive succinimide esters, cyclic thio esters or anhydrides may be used to introduce carbonyl functions which may react with the lysine amino group or to introduce a thiol reactive group such as those discussed herein. Alternatively, carbonyl functions may be activated with carbodimides to form carboxamide bonds or the lysine amino group may be reacted with methyl amidate esters to form an amidinium bond. One particular utility of the present invention is its application to in vitro or in vivo diagnostics. For example, in vitro detection of a particular antigen may be achieved by exposing a diagnostic sample to a conjugate of the present invention comprising a targetting moiety such as an antibody capable of binding to the antigen and subsequent exposure to a prodrug such as a methotrexate prodrug of the present invention, which may be catalysed to methotrexate by the enzyme of the conjugate if antigen is present and conjugate is bound to the antigen. Prodrug should be added after unbound conjugate has been removed. Catalysed prodrug such as MTX may be detected by standard HPLC analysis, or by the coupled spectrophotometric assay described herein. In vivo diagnostic applications of the present invention include its ability to be used in tumour imaging. Particularly preferred conjugates for use in this application include those comprising an antibodies to a tumour associated antigen and a mutant enzyme capable of catalysing to methotrexate, an MTX-prodrug labelled in the benzene ring with 1311. Such a prodrug should be one which is otherwise refractory to endogenous catalysis. Conjugate and prodrug may be administered in a manner analogous to the therapeutic application of the invention and detection of the location of catalysed prodrug i.e. I *- 1 MTX achieved using standard equipment for detection of radioisotopic decay. Salts of prodrugs of the present invention include pharmaceutically acceptable base salts, derived from an appropriate base, such as alkali metal (e.g. sodium), alkaline earth metal (e.g. magnesium) salts, ammonium and NX+4 (wherein X is Cj_4 alkyl) salts. Suitable pharmaceutically acceptable salts also include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, salicylic, p-toluene-sulfonic, tartaric, citric, acetic, trifluoroacetic methanesulfonic, formic, succinic, naphthalene-2-sulfonic, isethionic, lactobionic and benzenesulfonic. The pharmaceutically acceptable prodrug salts may be prepared in conventional manner for example by treatment with the appropriate base or acid. In a method of treatment according to the present invention the means of administration of conjugate and of prodrug will depend upon the disease and its severity and will be at the discretion of the practising physician. Both the conjugate and prodrug may be administered by the conventional methods well known to those skilled in the art including intraveneously, intraperitoneally, intralymphatically, subcutaneously, intradermally, intramuscularly, orally or in the case of a tumour by direct injection into the tumour. Both the prodrug and the conjugate will preferably be administered intravenously as a bolus or by continuous infusion. Preferably the dose of conjugate administered is in the range 0.1 to 100 mg per kg of bodyweight per day and most preferably between 1.0 and 40 mg per kg of bodyweight per day. The preferred dose of the prodrug administered is in the range 0.01 to 100 mg per kg of bodyweight per day and preferably between 1.0 and 50 mg per kg of bodyweight per day. It is preferred that the conjugate of targetting molecule and mutant enzyme be administered to the patient prior to administration of the prodrug. The period between administration of conjugate and prodrug must be sufficient to permit targetting of the conjugate to the targetted cells and effective removal from the system of excess conjugate which has not bound to the targetted cells. Unbound conjugate must be effectively removed to avoid activation of the subsequently administered prodrug by unbound conjugate present at sites distant from the target cells. The period between administration of conjugate and prodrug is preferably between 0.5 days and 7 days but may be reduced by use of any known technique for the accelerated clearance of such conjugates from the system; particularly by delivery of an effective amount of anti- enzyme antibody, which binds to the mutant enzyme and in turn accelerates elimination, or by extra corporeal circulation (Nilsson, I M, et al, Plasma Ther. Transfus Technol. 1984, 5, 127; Wallmark, A, et al, Artificial Organs. 1984, 8, 72) involving removal of the unbound conjugate by methods such as passage through a column of an appropriate affinity matrix. It may also be desirable to couple galactosyl residues to the conjugate to facilitate unbound conjugate clearance via hepatocyte lectins. Conditions susceptible to therapy according to the present invention will clearly include those for which there exists a known chemotherapeutic agent and a corresponding prodrug thereof as hereinbefore defined and which presents a pathology specific feature permitting binding of a conjugate of the present invention. Such conditions include neoplastic and non-neoplastic cellular transformation, autoimmune disease, inflammatory disease and viral, bacterial, fungal, mycoplasmal or parasitic infection. A method of treatment according to the present invention may further comprise the co- administration of a number of agents capable of increasing the efficiency of the therapy. For example, interferons are known to increase the level of expression of certain potential ADEPT target antigens and therefore administration of an appropriate interferon before administration of conjugate may increase level of conjugate binding to target cells. It is further preferred that a combination of a prodrug of a purine synthesis inhibitor such as an inhibitor of glycineamide ribonucleotide transformylase (GAR TFase) or a prodrug of a pyrimidine synthesis inhibitor such as an inhibitor of thymidine synthase (TS) be administered in combination with a prodrug of a dithydrofolate (DHFR) inhibitor as part of such therapy (Galivan, J. et al, J. Biol. Chem. 264, 10685, 1989, Ferguson, K. et al Cancer Chemother. Pharmacol. 23 173, 1989). It is further preferred that a combination of a prodrug of purine synthesis inhibitor such as an inhibitor of GAR TFase or a prodrug of a pyrimidine synthesis inhibitor such as an inhibitor of TS be administered in combination with DHFR inhibitor, or that a combination of a purine synthesis inhibitor such as an inhibitor of GAR TFase or a pyrimidine synthesis inhibitor such as an inhibitor of TS be administered in combination with a prodrug of a DHFR inhibitor as part of such therapy. A further aspect of the present invention comprises a method for the potentiation of any antiviral compound, the efficacy of which may be augmented by elevation of intracellular kinase activity. According to a preferred embodiment of this further aspect of the present invention, a conjugate of the present invention comprising a targetting molecule capable of directing conjugate to a virally-infected cell and a prodrug of a folate antagonist which may be one or more of a thymidylate synthase (TS) inhibitor, dihydrofolate reductase (DFHR) inhibitor or GAR transformylase inhibitor is used to deliver high doses of folate antagonist directly to the virally-infected cells of a patient who is undergoing therapy. As viral infection is associated with increased levels of DNA synthesis, it is proposed that the delivery of an inhibitor of DNA synthesis in combination with such an antiviral may prove therapeutic as the inhibition of DNA synthesis will increase the level of intracellular kinases available for phosphorylating the antiviral compound. In a preferred embodiment of this further aspect of the invention, the anti- viral is an anti- herpes compound selected from acyclovir (GB 1523865), valacyclovir (EP308065), famciclovir EP 182024), penciclovir (EP141927), ganciclovir (EP0072027), foscarnet (GB 1585615), iododeoxyuridine, 5-bromovinyl-2'-deoxyuridine (GB1601020), arabino- furanosyl-5-bromovinyl-2'-deoxyuridine (EP0031128), benzimidazole nucleosides, l-(β- D-arabinofuranosyl)-5-(l-propynyl)uracil (EP272065) and 2'-deoxy-5-ethyl-β-4'- thiouridine (EP409575), or is an anti-HIN compound selected from 2',3'-dideoxyinosine (EP206497), 2',3'-dideoxycytidine (J. Org. Chem. 32(3) 817-818 (1967)), (2R,5S)-5- fluoro- 1 -(2-(hydroxymethyl)- 1 ,3-oxathiolan-5-yl)cytosine (US5210085), (2R,5 S)- 1 -(2- (hydroxymethyl)-l,3-oxathiolan-5-yl)cytosine (EP0382526), 5-chloro-2',3'-dideoxy-3*- fluorouridine (EP0305117) and (lS,4R)-4-(2-amino-6-(cyclopropylamino)-9H-purin-9- yl)-2-cyclopentene-l -methanol (EP0434450) or is the anti-influenza compound 9-(2- Deoxy-2-fluoro-β-D-ribofuranosyl) guanine) (EP0417999). In the most preferred embodiment of this further aspect of the invention, the antiviral compound is an anti-HIN compound such as zidovudine (EP 196185), (Retrovir: registered trade mark of The Wellcome Foundation Limited), the conjugate comprises an antibody against an HIN-associated antigen such as GP120 and the prodrug is a prodrug of a TS inhibitor. The present invention provides for the use of a novel prodrug of the present invention as hereinbefore defined, or a pharmaceutically acceptable salt, solvate, N-oxide or physiologically functional derivative thereof, in therapy or in the preparation of a medicament for use in the treatment of any of the conditions hereinbefore described. Also provided is the use of a mutant enzyme of the present invention, or a conjugate thereof as hereinbefore described, in therapy or in the preparation of a medicament for use in the treatment of any of the conditions hereinbefore described. Pharmaceutical formulations for mutant enzyme, antibody and prodrug may each include but are not limited to solutions, suspensions, tablets, powders, and other formulations known to those skilled in the art. The formulations may contain pharmaceutically acceptable stabilisers and carriers including human serum albumin or other proteins, pH buffering agents, and physiological or other nontoxic salts as well as non toxic solubilisers known to those skilled in the art. Preferably a pharmaceutical formulation of mutant enzyme and antibody will comprise conjugate in a sterile solution. A pharmaceutical formulation of prodrug will preferably comprise a sterile solution or a solid. A yet further aspect of the present invention comprises a kit comprising a pharmaceutical formulation of conjugate and a pharmaceutical formulation of prodrug which may be converted to drug by that conjugate. A yet further aspect of the present invention comprises the combination of a prodrug and a conjugate of the present invention as hereinbefore defined. A yet further aspect of the present invention comprises the use of a novel prodrug as hereinbefore defined but excluding the previously stated proviso, for use in the preparation of a medicament for use in the treatment of any of the conditions hereinbefore described. A yet further aspect of the present invention comprises the use of a novel prodrug as hereinbefore defined but excluding the previously stated proviso, for use in the treatment of any of the conditions hereinbefore described. A yet further aspect of the present invention comprises the use of a prodrug in the preparation of a medicament for use in the treatment of a patient who has previously been administered with a conjugate of the present invention as hereinbefore defined. A further aspect of the invention comprises the cloned version of human carboxypeptidase A2 described herein as well as the DNA encoding carboxypeptidase A2. A further aspect of the invention comprises any DNA sequence encoding a mutant enzyme for use according to the present invention and plasmids, expression vectors, and cell lines containing such DNA. Preferably such DNA encodes mutant carboxypeptidase Al or A2 mutants as herein described and most preferable mutant carboxypeptidase Al or A2 wherein position 268 is glycine. A further aspect of the invention comprises DNA sequence encoding a conjugate according to the present invention and plasmids, expression vectors, and cell lines containing such DNA. A yet further agreed aspect of the invention is a method of converting a prodrug to a drug which is cytotoxic to cells. All references to alternatives herein, such as alternative patterns of chemical substitutions, shall be construed as inclusive and not as exclusive. As used herein the term "halo" means fluoro, chloro, bromo or iodo. The terms "aryl" means phenyl, naphthyl or anthracenyl optionally substituted by one or more halo, hydroxy, nitro, cyano, trifluoromethyl, Cι_6 alkyl, C\. alkoxy or amino optionally substituted with C\.^ alkyl. The term "heteroaryl" means an aromatic monocyclic or bicyclic fused ring system comprising 5 to 10 carbon atoms wherein one or more ring atoms are independently selected from nitrogen, oxygen or suffer. "Carboxamide" groups can be unsubstituted or substituted C\. alkyl. A mutant mammalian enzyme as used herein shall be taken to be any enzyme with a sequence differing by at least one amino acid from the amino acid sequence or sequences of that enzyme in the mammal to which the therapy is applied. Preferably the mammal to which the therapy is applied is a human patient and preferably the amino acid sequence of the mutant mammalian enzyme does not differ by more than 15% from the amino acid sequence or sequences of that enzyme in the mammal to which the therapy is applied. All references hereinbefore and in the Claims hereinafter to residue numbers of amino acids relate to the residue number indicated in the Tables contained herein and not to the residue numbers given in the Sequence Listing herein which follows a different numbering convention. Experimental Procedures General Comments All solvents were reagent grade and were used without further purification. Anhydrous solvents were dispensed from Aldrich Sure Seal bottles using a dry syringe. Chemicals are reagent grade and were used without purification. The full name and address of the suppliers of the reagents is given when first cited. Thereafter, an abbreviated name is used. ^H-NMR spectra were obtained using Narian XL-200 or XL-300 spectrometers at 200 and 300MHz respectively. Chemical shifts are expressed in ppm downfield from tetramethylsilane. Spectral data are tabulated in order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), number of hydrogens, coupling constant(s) in Hertz, descriptor. Chemical ionization (CI) mass spectra were performed by Oneida Research Services, Inc., Whitesboro, ΝY 13492. Atmospheric pressure chemical ionization (APCI) mass spectra were obtained using a Fisons platform mass spectrometer. Fast atom bombardment (FAB) mass spectra were obtained using a NG Analytical 70SQ spectrometer. Ion spray mass spectra were obtained using a Sciex API m spectrometer. Elemental analyses were performed by Atlantic Microlabs, Inc., Atlanta, GA 30366. Reverse phase analytical HPLC's were run on C18 analytical columns using a Waters HPLC system consisting of a 600E System Controller, a 715 UltraWisp sample processor, and a 991 Photodiode Array Detector. Semi-preparative HPLC's were run on a Regis column (C18, Prep-100-60-ODS-FEC, 10mm packing, 25cm x 21.1mm i.d.) using a similar system equipped with a manual injector. Abbreviations used are: grams (g), milliliters (mL), liters (L), hours (h), minutes (min), room temperature (RT), calculated (calcd.), decomposition (dec.), molecular weight (mw), tetrahydrofuran (THF), dimethyl formamide (DMF), dimethyl sulphoxide (DMSO), l,3-dimethyl-3,4,5,6-tetrahydro-2(lH)-pyrimidinone (DMPU), l-ethyl-3- (dimethylaminopropyl)carbodiimide hydrochloride (EDC), dicyclohexylcarbdiimide (DCC), diethyl cyanophosphonate (DECP), (benzyloxy)carbonyl (Cbz), and trifluoroacetic acid (TFA). Some examples prepared were very tenacious of water and or other hydroxylic solvents (e.g. DMF), and or were obtained as whole or partial salts of organic or inorganic acids (e.g. trifluoroacetic acid, hydrochloric acid). Such addends are indicated in the analytical data for appropriate examples; although these addends may not be indicated as part of the compound name, their presence in the isolated products is noted by the inclusion of salt and hydration data in the molecular formulas and analytical data in appropriate examples. Example 1 : 2-Fluoro-4-nitrobenzoic acid A 3L 3 -necked flask equipped with a magnetic stirrer was charged with 25.00 g of 2-fluoro-4-nitrotoluene (Aldrich Chemical Co., Milwaukee, WI, 53233), 1.65 L of IN NaOH, and 25.00 g of KMnθ4. The resulting mixture was heated to 95°C with stirring. Additional 25.00 g portions of KMnθ4 were added after lh and 2h. After stirring at 95°C for an additional hour the reaction mixture was cooled to RT and filtered through celite to remove Mnθ2- The filtrate was concentrated in vacuo to 500 mL and acidified to pH 2 by addition of concentrated H2SO4. A yellow-orange precipitate formed which was collected by vacuum filtration. The crude solid was dissolved in 300 mL of IN aqueous NaOH, acidified to pH 2 and the mixture extracted with CH2CI2 (4 x 150 mL). The combined CH2CI2 extracts were washed with 0.1 N aqueous HCl (2 x 100 mL), dried over anhydrous Na2SO4, and concentrated in vacuo to afford 12.44 g (42%) of 2-fluoro-4-nitrobenzoic acid as a yellow crystalline solid, m.p. 165-167°C. Example 2: Ethyl 2-fluoro-4-nitrobenzoate To a dry 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 3.00 g of 2-fluoro-4-nitrobenzoic acid, 3.00 g of NaHCO3, and 20 mL of anhydrous dimethylacetamide. The suspension was treated with 3.50 mL of ethyl iodide and stirred at RT under N 2 for 18h. The reaction mixture was filtered to remove solids and the filtrate concentrated in vacuo to give an orange residue which was dissolved in 100 mL of CH 2 C1 2 . The solution was washed with saturated aqueous NaHCO3 (3 x 50 mL), water (1 x 60 mL), and dried over anhydrous Na 2 SO4. Removal of drying agent by filtration and passage through a silica gel plug (3 x 5 cm) gave a light yellow solution which was concentrated in vacuo to afford 3.10 g (90%) of ethyl 2-fluoro-4- nitrobenzoate as a light yellow crystalline solid, m.p. 70-73°C. Example 3 : Ethyl 4-amino-2-fluorobenzoate To a 250 mL round bottomed flask equipped with a magnetic stirrer were added 3.00 g of ethyl 2-fluoro-4-nitro-benzoate and 100 L of MeOH. The solution was deoxygenated by bubbling nitrogen through for 10 min and then treated with 2.00 g of Raney nickel (Raney 3200, Davison Chemical, Chattanooga, TN 37406) which had been washed with water and EtOH. The solution was subjected to hydrogenation at 1 at with stirring for 40 min. The flask was purged with nitrogen and the catalyst was removed by filtration. The filtrate was concentrated in vacuo to afford 2.40 g (93%) of ethyl-4-amino-2-fluorobenzoate as a tan crystalline solid, m.p. 110-112°C. Example 4: 9-Bromomethyl-3-methylbenzoff.quinazolin-lf2H.-one To a 3L 3-necked flask equipped with a magnetic stirrer, a nitrogen inlet, and a reflux condenser were added 1.4 L of 1,2-dichloroethane. The solvent was heated to reflux and 10.00 g of 3,9-dimethylbenzo(f)-quinazolin-l(2H)-one (prepared according to the method described in International Patent Application WO 91/19700) were slowly added. This was followed by addition of 8.73 g of N-bromosuccinimide (Aldrich) and 1.00 g of benzoyl peroxide (Aldrich). After heating at reflux for 2h the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was slurried with 60 mL of EtOH and the solid was collected by vacuum filtration. The product was washed with 50 mL of EtOH and dried in vacuo to afford 12.39 g of a light tan solid, m.p. >200°C. Analysis by -1H-ΝMR indicated a mixture consisting of 80% of 9- bromomethyl-3-methylbenzo(f)quinazolin-l(2H)-one and 20% of starting material. The material was carried on to the next step without further purification. JH-NMR: (300 MHz, DMSO-d6) δ 9.86 (s, IH, aromatic CH), 8.28 (d, IH, J=9.0, aromatic CH), 8.06 (d, IH, J=8.4, aromatic CH), 7.72 (d, IH, J=8.2, aromatic CH), 7.65 (d, IH, J=8.8, aromatic CH), 4.94 (s, 2H, ArCH 2 Br), 2.49 (s, 3H, CH3). Example 5: Ethyl 4-fffl.2-dihydro-3-rnethyl-l-oxobenzoff.quinazolin-9- yDmethyDamino ' .-2-fluoroberιzoate To a 500 mL 3-neck flask equipped with a magnetic stirrer, a nitrogen inlet, and a reflux condenser were added 5.00 g of 9-bromomethyl-3-methylbenzo(f)quinazolin-l(2H)-one, 3.02 g of ethyl 4-amino-2-fluorobenzoate, 4.17 g of NaHCO3, and 42 mL of anhydrous DMF. The reaction mixture was heated to 100°C with stirring under nitrogen for 1.5h. After cooling to RT the mixture was filtered to remove solids and the crude product was deposited on 30 g of silicon gel by concentrating the filtrate in vacuo in the presence of silica gel. The product silica gel mixture was applied to a column (360 g Siθ2) and purified by flash chromatography using MeOH-EtOAc gradient elution (EtOAc -* 2% MeOH-EtOAc). This afforded 3.50 g (65%) of ethyl 4-(((l,2-dihydro-3-methyl-l- oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoate as a white powder, m.p. >200°C. JH-NMR: (300 MHz, DMSO-d6) δ 12.54 (s, IH, NH), 9.85 (s, IH, aromatic CH), 8.23 (d, IH, J=8.7, aromatic CH), 8.01 (d, IH, J=8.2, aromatic CH), 7.69-7.50 (m, 3H, aromatic CH), 6.52 (dd; IH; J=1.8, 8.9; aromatic CH), 6.40 (dd; IH; J=1.7, 14.7; aromatic CH), 4.58 (d, 2H, J=5.6, ArCH 2 N), 4.19 (q, 2H, J=7.0, ethyl CH 2 ), 2.44 (s, 3H, CH3), 1.24 (t, 3H, J=7.0, ethyl CH3). Example 6: 4-ffπ.2-Dihydro-3-methyl-l-oxobenzoff.quinazolin-9-yπmethyDamino'.-2- fluorobenzoic acid A 100 mL 3-necked flask equipped with a magnetic stirrer was charged with 0.200 g of ethyl 4-((( 1 ,2-dihydro-3 -methyl- 1 -oxobenzo(f)quinazolin-9-yl)methyl)amino)-2- fluorobenzoate, 18 mL of IN NaOH, and 20 mL of EtOH. After stirring overnight at RT, the solution was concentrated to 20 mL in vacuo and acidified to pH 2.5 by addition of concentrated HCl. The resulting precipitate was collected by vacuum filtration, washed with 15 mL of water, and dried in vacuo to afford 0.15 g (77%) of 4-(((l,2- dihydro-3 -methyl- l-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoic acid as a white powder, m.p. >250°C. Ϊ H-NMR: (300 MHz, DMSO-d6) δ 13.20-12.30 (br s, IH, COOH), 9.83 (s, IH, aromatic CH), 8.24 (d, IH, J=8.6, aromatic CH), 8.06 (d, IH, J=8.4, aromatic CH), 7.80-7.40 (m, 4H, aromatic CH), 6.51 (d, IH, J=7.9, aromatic CH), 6.37 (d, IH, J=14.2, aromatic CH), 4.60 (s, 2H, ArCH2N), 2.48 (s, 3H, CH3). Example 7: N-(fBenzyloxy ' .carbonylV5-O-ethyl-L-glutamic acid A 2L 3-necked flask equipped with a magnetic stirrer and a thermometer was charged with 25.0 g of glutamic acid γ-ethyl ester (Sigma Chemical Co., St. Louis, MΟ, 63178) and IL of distilled water. The solution was heated to 60°C with stirring and treated with 30 g of ΝaHCθ3 followed by 25 mL of benzyl chloroformate (Aldrich). The flask was allowed to cool to RT and stirring was continued for 2h. The mixture was extracted with ethyl ether (5 x 100 mL) and the ether extracts were discarded. The aqueous phase was acidified to a congo red end point by addition of concentrated aqueous HCl and the resulting emulsion was extracted with CH Cl2 (5 x 100 mL). The combined CH 2 C1 2 extracts were washed with 0.05 N aqueous HCl (2 x 100 mL), dried over anhydrous Na2SO4, and concentrated in vacuo to a volume of 50 mL. The solution was chilled in an ice water bath and triturated with addition of 200 mL of pentane. The resulting solid was collected by vacuum filtration and dried in vacuo to afford 37.0 g (83%) of N-((benzyloxy)carbonyl)-5-0-ethyl-L-glutamic acid as a white powder, m.p. 82-84°C. Example 8 : Ethyl 3 -f 1 -naphthylVL-alaninate To a 250 mL 3-necked flask equipped with a magnetic stirrer and a reflux condenser were added 3.00 g of 3-(l'-naphthyl)-L-alanine (Schweizerhall, Inc., Piscataway, ΝJ, 08854) and 75 mL of absolute EtOH. The suspension was acidified by bubbling HCl gas through until a clear solution was obtained and the solution was heated at reflux with stirring for 5h. The solution was cooled to RT and concentrated in vacuo to give a white solid, which was suspended in 100 mL of 5% aqueous ΝaHCθ3 and extracted with EtOAc (3 x 60 mL). The combined EtOAc extracts were washed with 5% aqueous NaHCO3 (3 x 50 mL), water (1 x 50 mL), and dried over anhydrous Na2SO4. The drying agent was removed by filtration and the filtrate concentrated in vacuo to give an oil which was dissolved in 40 mL of ethyl ether. The ether solution was treated with 13.9 mL of IN ethereal HCl and the resulting precipitate was collected by vacuum filtration and dried in vacuo to afford 3.70 g (95%) of ethyl 3-(l-naphthyl)-L- alaninate HCl as a white powder, m.p. 182-184°C. Example 9: Ethyl N-ffbenzyloxy.cari?onvO-5-O-ethyl-L-glutam-l-yl-3-f 1-naphthylVL- alaninate A 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 3.70 g of ethyl 3-(l-naphthyl)-L-alaninate*HCl, 4.09 g of N-((benzyloxy)carbonyl)-5-Ο-ethyl-L-glutamic acid, 60 mL of CH 2 C1 2 , and 1.45 mL of N-methylmorpholine. The mixture was chilled to 0°C in an ice bath, treated with 2.66 g of EDC (Aldrich) and stirred under nitrogen. After lh at 0°C the solution was allowed to warm to RT and stirring was continued for an additional 3h. The solution was concentrated in vacuo and the residue was dissolved in 100 mL of EtOAc. The EtOAc solution was washed with 10% aqueous citric acid (3 x 50 mL) followed by 5% aqueous ΝaHC03 (3 x 50 ml). The solution was dried over anhydrous Na 2 SO4, concentrated in vacuo to 20 mL. and triturated with addition of 50 mL of pentane. The resulting solid was collected by vacuum filtration and dried in vacuo to afford 5.80 g (82%) of ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-l-yl-3-(l-naphthyl)-L-alaninate as a white powder, m.p. 124-126°C. Example 10: Ethyl 5-O-ethyl-L-glutam-l-yl-3-fl-naphthvP-L-alaninate A 500 mL Parr bottle was charged with 0.19 g of 10% Pd(C) and 60 mL of absolute EtOH under nitrogen. The catalyst-solvent mixture was deoxygenated by bubbling nitrogen through for 5 minutes and then treated with 0.14 mL of acetyl chloride followed by 1.02 g of ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-l-yl-3-(l-naphthyl)-L- alaninate. The mixture was hydrogenated at 45 psi for 18h. The bottle was purged with nitrogen and the catalyst was removed by filtration through celite. The filtrate was concentrated in vacuo to afford 0.81 g (97%) of ethyl 5-0-ethyl-L-glutam-l-yl-3-(l- naphthyl)-L-alaninate HCl as a light yellow foam, m.p. 85-90°C (dec). Example 11 : Ethyl N-fN- 4-f f 1 ,2-dihvdro-3-methyl- 1 -oxobenzof f.quinazolin-9- yDmethyl)aminoy2-fluoroberιzoylV5-O-ethyl-L-glutam- 1 -yP-3-f 1 -naphthylVL-alaninate A 100 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 0.50 g of 4-(((l,2-dihydro-3-methyl-l-oxobenzo(f)quinazolin-9- yl)methyl)amino)-2-fluorobenzoic acid and 10 mL of DMPU (Aldrich). The mixture was alternately heated and sonicated to dissolve the solid and the resulting solution was stirred under nitrogen in the presence of 0.50 g of 3A molecular sieves for 18h. The mixture was cooled to 0°C in an ice bath and treated with 0.15 mL of Ν-methylmorpholine followed by 0.17 mL of isobutyl chloroformate. After stirring at 0°C for 15 min the reaction mixture was treated with a solution of 0.69 g of ethyl 5-O- ethyl-L-glutam-l-yl-3-(l-naphthyl)-L-alaninate*HCl and 0.17 mL of N-methylmorpholine in 4 mL of anhydrous DMF. The reaction was maintained at 0°C for lh and was then allowed to warm to RT and stirred for an additional 4h. The mixture was filtered to remove solids and the filtrate was mixed with 150 mL of water. The resulting crude solid was collected by filtration, dried in vacuo, and subjected to flash chromatography on 200 g of silica gel (1% MeOH/EtOAc → 5% MeOH/EtOAc) to afford 0.40 g (40%) of ethyl N-(N-(4-((( 1 ,2-dihydro-3 -methyl- 1 -oxobenzo(f)quinazolin-9-yl)methyl)amino)- 2-fluorobenzoyl)-5-O-ethyl-L-glutam-l-yl)-3-(l-naphthyl)-L-alaninate as a white powder, m.p. 225°C (dec). iH-NMR: (200 MHz, DMSO-d6) δ 12.55 (s, IH, benzaquinazoline NH), 9.87 (s, IH, aromatic CH), 8.63 (d, IH, J=7.5, amide NH), 8.24 (d, IH, J=8.3, aromatic CH), 8.09 (d, IH, J=7.7, aromatic CH), 8.03 (d, IH, J=8.0, aromatic CH), 7.92 (d, IH, J=7.1, aromatic CH), 7.79 (t, IH, J=5.6, 4-aminobenzoyl NH), 7.68-7.30 (m, 9H, aromatic CH, amide NH), 6.56 (d, IH, J=7.9, aromatic CH), 6.42 (d, IH, J=14.4, aromatic CH), 4.68-4.43 (m, 4H, benzoquinazoline-9-CH2, glu methine, naphthylala methine), 4.10-3.92 (m, 4H, ethyl CH2), 3.63-3.30 (m, 2H, naphthyl-CH2), 2.45 (benzoquinazoline CH3), 2.29 (m, 2H, glu 4-CH 2 ), 2.07-1.74 (m, 2H, glu 3-CH 2 ), 1.14(t, 3H, J=7.1, ethyl CH3), 1.03 (t, 3H, J=7.1, ethyl CH3). Example 12 : N-f N-(4-f f 1.2-Dihydro-3-methyl- 1 -oxobenzof f.quinazolin-9- yπmethyDaminoy 2-fluorobenzoyl'. -L-glutam- 1 -yl V3 -f 1 -naphthylVL-alanine To a 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 0.15 g of ethyl N-(N-(4-(((l, 2-dihydro-3-methyl-l-oxobenzo(f)quinazolin-9- yl)methyl)amino)-2-fluorobenzoyl)-5-O-ethyl-L-glutam- 1 -yl)-3-( 1 -naphthyl)-L-alaninate, 8 mL of 1 : 1 THF : H 2 O, and 20 mg of LiOH H 2 O. The mixture was stirred at RT under nitrogen. After lh analysis of the solution by tic (SiO 2 , 3% MeOH/CH 2 C_2) showed no remaining starting material and a new component at RρO.0. The solution was acidified to pH 5 by addition of IN aqueous HCl and the resulting white suspension was concentrated in vacuo to dryness. Analysis of the residue by reverse phase HPLC (C18, 65 : 35 : 0.1 H 2 O : MeCΝ : TFA) indicated a major component (95%) at k'=3.17 and a minor component (5%) at k'=4.90. The crude product was purified by semi- preparative reverse phase HPLC using gradient elution (C18, 70 : 30 : 0.1 _-£ 65 : 35 : 0.1 H 2 O : MeCΝ : TFA over 10 min). Pure fractions (as determined by analytical HPLC) were combined, concentrated to 20 mL in vacuo and lyophilized to afford 76 mg (50%) ofN-(N-( 4 -((( 1 - -dihydro-3-methyl-l-oxobenzo(f)-quinazolin-9- yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-l-yl)-3-(l-naphthyl)-L-alanine as an off white powder, m.p. 180°C (dec). HPLC: one peak on C18, k'=3.53, 65 : 35 : 0.1 H 2 O:MeCN:TFA, flow rate = 1 mlJmin. ^- MR: (300 MHz, DMSO-dό) δ 13.30-12.20 (br m, COOH), 9.82 (s, IH, aromatic CH), 8.44 (d, IH, J=8.1, amide NH), 8.26 (d, IH, J=8.9, aromatic CH), 8.11 (d, IH, J=8.3, aromatic CH), 8.03 (d, IH, J=8.3, aromatic CH), 7.84 (d, IH, J=8.2, aromatic CH), 7.73 (d, IH, J=7.8, aromatic CH), 7.67-7.27 (m, 9H, aromatic CH, amide NH), 6.53 (d, IH, J=8.5, aromatic CH), 6.38 (d, IH, J=14.7, aromatic CH), 4.63-4.38 (m, 4H, benzoquinazoline-9-CH2, glu methine, naphthylala methine), 3.55 (m, IH, naphthyl- CH 2 ), 3.27 (m, IH, naphthyl-CH 2 ), 2.43 (s, 3H, benzoquinazoline CH3), 2.18 (m, 2H, glu 4-CH 2 ), 2.00-1.70 (m, 2H, glu 3-CH 2 ). Elemental Analysis: Calcd. for C39H34N5O7F* 1.5 H 2 O 0.5 TFA (MW 787.76): C, 60.99; H, 4.80; N, 8.89. Found: C, 60.96; H, 4.82; N, 8.98. Mass Spectrum: (FAB) 704 (M + H) + , 613, 489, 371, 309. UN Spectrum: (pH 7 Buffer) λ ma χ (ε) 223.4 (82600), 266.4 (53100), 348.6 (5500). λmin (ε) 243.2 (23500), 340.2 (3430). Example 13: Ethyl N-f4-fff2.4-diamino-6-pteridinyl.methyπmethylamino ' .benzoyπ-5-O- ethyl-L-glutam- 1 -yl-3 -f 1 -naphthyl VL-alaninate To a solution of 0.24 mL of DECP (Aldrich) and 0.22 mL of Et3Ν in 20 mL of anhydrous DMF in a 100 mL 3-necked flask were added 0.20 g of 4-(N-(2,4-diamino-6- pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich Chemical Co., Milwaukee, WI, 53233) and 0.04 mL of Et3Ν dissolved in 3 mL of DMPU. The solution was stirred at RT under nitrogen for 3h and treated with a solution of 0.23 g of ethyl 5-O-ethyl-L-glutam-l-yl-3-(l-naρhthyl)-L-alaninate*HCl and 0.15 mL of Et3N in 2 mL of anhydrous DMF. After stirring for 70h the reaction mixture was concentrated in vacuo to 3 mL and treated with 60 mL of ethyl ether to precipitate the crude product as a tacky orange solid. The ether was decanted and the product dissolved in 60 mL of CHCI3. The solution was extracted with 1% aqueous NH4OH, dried over anhydrous Na2SO4 and concentrated in vacuo to dryness. The yellow-orange residue was purified by flash chromatography on 150 g of silica gel (7 : 7 : 1 CH2CI2: acetone : MeOH) to afford 0.26 g (70%) of ethyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-l-yl-3-(l-naphthyl)-L- alaninate as a yellow powder, m.p. 135°C (dec). ϊϊfc M : (200 MHz, DMSΟ-d6) δ 8.59 (s, IH, pteridinyl-7-CH), 8.45 (d, IH, J=7.2, amide ΝH), 8.10 (d, IH, J=8.0, aromatic CH), 8.04 (d, IH, J=8.2, aromatic CH), 7.92 (d, IH, J=6.8, aromatic CH), 7.83-7.45 (m, 6H, aromatic CH, ΝH2), 7.41-7.23 (m, 2H, aromatic CH), 6.84 (d, 2H, J=8.8, aromatic CH), 6.65 (br s, 2H, NH2), 4.81 (s, 2H, pteridinyl-CH2), 4.63-4.39 (m, 2H, glu methine, naphthylala methine), 4.13-3.91 (m, 4H, ethyl CH2), 3.63-3.32 (m, 2H, naphthyl-CH 2 ), 3.24 (s, 3H, N-CH3), 2.34 (t, 2H, J=7.6, glu-4-CH2), 2.10-1.80 (m, 2H, glu-3-CH2), 1.17 (t, 3H, J=7.1, ethyl-CH3), 1.03 (t, 3H, J=7.1, ethyl-CH3). Example 14 : N- 4- f f 2.4-Diamino-6-pteridinyl'.methvπmethylamino')benzoyπ-L-glutam- 1 -yl-3-f 1 -naphthvD-L-alanine To a 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 0.13 g of ethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5- O-ethyl-L-glutam-l-yl-3-(l-naρhthyl)-L-alaninate, 10 mL of EtOH and 15 mL of 0.2 Ν ΝaOH. The resulting solution was stirred at RT under Ν 2 for 2.5h, acidified to pH 5 by addition of IN aqueous HCl, and concentrated in vacuo to dryness. Analysis of the yellow solid by reverse phase HPLC (C18, 70 : 30 : 0.1 H2O : MeCN : TFA) indicated a major component (93%) at k'=4.3 and four minor components at k'=6.8, 3.5, 2.9 and 1.9. A 70 mg portion of the crude product was purified by semi-preparative reverse phase HPLC (C18, 70 : 30 : 0.1 H 2 O : MeCN : TFA). Pure fractions (as determined by analytical HPLC) were combined, concentrated in vacuo to 20 mL and lyophilized to afford 36 mg (53%) of N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3-( 1 -naphthyl)-L-alanine as a yellow powder, m.p. 175°C (dec). HPLC: one peak on C18, k'=3.23, 70 : 30 : 0.1 H 2 O : MeCΝ : TFA, flow rate=l mlJmin. Ϊ H-ΝMR: (300 MHz, DMSO-dό) δ 13.00-11.95 (br m, 2H, COOH), 8.65 (s, IH, pteridinyl-7-CH), 8.45 (br, IH, NH 2 ), 8.36-8.18 (br, IH, NH 2 ), 8.25 (d, IH, J=7.5, amide NH), 8.10 (d, IH, J=8.6, aromatic CH), 7.96 (d, IH, J=7.8, aromatic CH), 7.90 (d, IH, J=7.7, aromatic CH), 7.70 (m, 3H, aromatic CH), 7.60-7.45 (m, 3H, aromatic CH, NH 2 ), 7.39-7.15 (m, 3H, aromatic CH, amide NH, NH2), 6.81 (d, 2H, J=8.6, aromatic CH, 4.84 (s, 2H, pteridinyl-CH2), 4.51 (m, IH, naphthylala methine), 4.42 (m, IH, glu methine), 3.57 (m, IH, naphthylala-CH2), 3.28 (m, overlapping H2O peak, naphthylala CH2), 3.25 (s, 3H, N-CH3), 2.23 (t, 2H, J=7.5, glu-4-CH2), 2.02-1.72 (m, 2H, glu-3-CH2). Elemental Analysis: Calcd. for C33H33N9O6- 1.5 H2O O.5 TFA (MW 735.72): C, 55.51; H, 5.00; N, 17.13. Found: C, 55.65; H, 5.09; N, 16.92. Mass Spectrum: (FAB) 652 (M + H) + , 581, 461, 371, 309. UN Spectrum: (pH 7 Buffer) λmax (ε) 224.3 (74700), 260.3 (24900), 295.9 (24100), 372.5 (7150). λmin (ε) 242 * 3 (15200), 271.0 (20500), 345.2 (5550). Example 15: N*--ffBenzyloxyte_u onylV5-O-methyl-L-glut__mic acid To a IL 3-necked flask equipped with a thermometer and a magnetic stirrer were added 4.90 g of L-glutamic acid γ-methyl ester (Sigma) and 200 mL of water. The solution was heated to 60°C, treated with 6.40 g of ΝaHC03 followed by 5.20 mL of benzyl chloroformate (Aldrich) and allowed to cool to RT with vigorous stirring. After 4h the reaction mixture was extracted with ethyl ether (4 x 100 mL) and the ether extracts were discarded. The aqueous solution was acidified to a congo red endpoint by addition of concentrated HCl. An oily emulsion resulted which was extracted with CH2C1 2 (4 x 60 mL). The combined organic extracts were washed with 0.05 N aqueous HCl (2 x 60 mL), dried over anhydrous Na 2 SO4 and concentrated in vacuo to afford 8.23 g (92%) of N-((benzyloxy)carbonyl)-5-O-methyl-L-glutamic acid as a white crystalline solid, m.p. 61-63°C. Example 16: Glycine tert-butyl ester-benzophenone imine A 500 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 10.00 g of glycine-tert-butyl ester-HCl (Sigma) and 200 mL of anhydrous CH 2 C1 2 . The solution was treated with 10.80 g of benzophenone imine (Aldrich) and stirred at RT under nitrogen for 24h. A fine white precipitate of ΝH4CI formed during the reaction. The mixture was concentrated in vacuo to dryness to give a white solid which was suspended in 250 mL of ethyl ether and washed with water (3 x 200 mL) to remove NH4CI. The ether solution was dried over anhydrous MgSO4 and concentrated in vacuo to afford a white solid. The material was dissolved in 30 mL of CH2CI2 and triturated with addition of 150 mL of pertane. A white crystalline solid resulted which was collected by filtration, dried in vacuo, and identified as glycine tert-butyl ester- benzophenone imine by -NMR. yield=15.0 g (85%), m.p. 110 -112°C. Example 17: 2-Iodobenzyl bromide To a dry 100 mL 3 -necked flask equipped with a nitrogen inlet and a magnetic stirrer were added 5.00 g of 2-iodobenzyl alcohol (Aldrich) and 20 mL of anhydrous CH2CI2. The resulting suspension was cooled to 0°C and treated with 0.71 mL of PBr3 (Aldrich) by dropwise addition over a 2 minute period. The solid slowly dissolved giving a light yellow solution which was stirred under nitrogen at 0°C for 2h and then allowed to warm to RT with continued stirring for 18h. The solution was concentrated in vacuo to dryness and the residue was dissolved in 60 mL of ethyl ether. The ether solution was washed with ice cold 5% aqueous NaHCO3 (3 x 50 mL), dried over anhydrous Na2SO4, and concentrated in vacuo to afford 5.65 g (89%) of 2-iodobenzyl bromide as a white crystalline solid, m.p. 52-54°C. Example 18: 2-Iodo-L-phenylalanine tert-butylester-benzophenone imine A 500 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 20.00 g of glycine tert-butyl ester-benzophenone imine, 24.13 g of 2- iodobenzyl bromide, 2.85 g of N-benzylcinchonidinium chloride (Fluka Chemical Corp., Ronkonkoma, ΝY, 11779), and 105 mL of CH2CI2. The resulting solution was treated with 110 mL of 50% (W/W) aqueous ΝaOH and the mixture was stirred vigorously at RT under nitrogen. After 24h, tic (silica gel, 7 : 1 hexane : EtOAc) indicated a major new component at Rf=0.65, unreacted starting material at Rf=0.85 and three minor components at Rf=0.20-0.45. The CH2CI2 phase was separated, washed with water (3 x 70 mL), dried over anhydrous Νa2Sθ4 and concentrated in vacuo to afford a thick pink oil. The crude product was subjected to flash chromatography on 550 g of silica gel eluting with 7 : 1 hexane : EtOAc (column was pretreated by passing through two column volumes of 0.5% Et3N in 7 : 1 hexane : EtOAc) to give 26.4 g (76%) of the desired enantiomer mixture as identified by -1H-NMR. The product was determined to be a mixture of two enantiomers by chiral HPLC (Pirkle phenylglycine, 99 : 1 hexane : isopropanol, flow rate=l mlJmin): k'=3.66 (76%), k'=3.28 (24%). The mixture was dissolved in 160 mL of hexane and allowed to crystallize at 4°C for 2 days. The solid was separated by filtration and dried in vacuo to afford 10.72 g (31%) of racemic material. The filtrate was concentrated in vacuo to give 15.72 g (45%) of enantioenriched material (2-iodo-L-phenylalanine tert-butyl ester-benzophenone imine, ee=86%) as a transparent, viscous oil. The product was used in subsequent steps without further purification. 1 IM_-MR: (200 MHz, CDCI3) d 7.72 (d, IH, J=7.8, aromatic CH), 7.60 (m, 2H, aromatic CH), 7.41-7.21 (m, 6H, aromatic CH), 7.19 (m, 2H, aromatic CH), 6.86 (m, IH, aromatic CH), 6.55 (d, 2H, J=6.6, aromatic CH), 4.34 (dd; IH; J=3.9, 9.6; methine), 3.48-3.21 (m, 2H, Ar-CH2), 1.47 (s, 9H, t-Bu). Mass Spectrum: (CI, CH4) 512 (M + H) + , 484, 456, 410, 384, 217. Example 19: 2-Iodo-L-phenylalanine A 500 mL round bottomed flask equipped with a reflux condenser and a magnetic stirrer was charged with 15.72 g of 2-iodo-L-phenylalanine tert-butyl ester-benzophenone imine (86% ee) and 165 mL of 6N aqueous HCl. The mixture was heated at reflux with stirring for 4h and cooled to RT to afford a mixture of white crystalline solid and an immicible organic phase (benzophenone). The mixture was partitioned between water (100 mL) and ethyl ether (70 mL). The aqueous phase was separated, washed with four additional 70 mL portions of ether and concentrated in vacuo to dryness to give 9.5 g (94%) of 2-iodo-L-phenylalanine HCl as a fluffy white solid, m.p. 240°C (dec). iH-NMR: (300 MHz, DMSO-dό) δ 8.90-8.23 (br s, 3H, -NH3 + ), 7.88 (d, IH, J=8.1, aromatic CH), 7.39 (m, 2H, aromatic CH), 7.04 (m, IH, aromatic CH), 4.06 (t, IH, J=7.4, methine), 3.23 (m, 2H, Ar-CH2). Example 20: Methyl 2-iodo-L-phenylalaninate To a 500 mL 3-necked flask equipped with a magnetic stirrer and a reflux condenser were added 5.00 g of 2-iodo-L-phenylalanine HCl and 100 mL of anhydrous MeOH. The mixture was stirred and acidified by bubbling HCl gas through for 3 min. A clear solution resulted which was heated at reflux for 5h. The solution was cooled to RT and concentrated in vacuo to dryness to give a white solid. The material was suspended in 120 mL of CH 2 C1 2 and washed with 5% aqueous NaHCO3 (4 x 80 mL). The CH 2 C1 2 solution was concentrated to a volume of 20 mL, diluted to 60 mL with ethyl ether and treated with 16 mL of 1M ethereal HCl. A white solid precipitated which was collected by vacuum filtration and dried in vacuo to afford 4.90 g (94%) of methyl 2-iodo-L- phenylalaninate HCl, m.p. 194-196°C. Example 21 : Methyl N-ffbenzyloxy1carbonyl -5-O-methyl-L-glutam- 1 -yl-2-iodo-L- phenylalaninate A dry 500 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 3.00 g of methyl 2-iodo-L-phenylalaninate HCl, 2.59 g of N- ((benzyloxy)carbonyl)-5-O-methyl-L-glutamic acid and 80 mL of CH 2 C1 2 . The mixture was cooled to 0°C in an ice water bath and was treated with 0.97 mL of Ν-methylmorpholine followed by 1.77 g of EDC (Aldrich). The reaction mixture was stirred at 0°C for lh and allowed to warm to RT with continued stirring for an additional 3h. The solution was diluted with 100 mL of CH 2 C_2, washed with 5% aqueous citric acid (3 x 70 mL), 5% aqueous ΝaHC03 (3 x 70 mL), and dried over anhydrous Na2Sθ4. Removal of the drying agent by filtration followed by in vacuo concentration of the filtrate afforded 4.30 g (84%) of an off-white crystalline solid, m.p. 110-112°C. Examination by *H-NMR indicated methyl N-((benzyloxy)carbonyl)-5-O-methyl-L- glutam-l-yl-2-iodo-L-phenylalaninate contaminated with approximately 4% of the (S-R)-diastereomer. The crude product was recrystallized twice from 1 : 1 EtOH-H2θ to give 3.47 g (68%) of pure (S,S)-diastereomer as a white crystalline solid, m.p. 114-116 0 C. Example 22: Methyl N-ffbenzyloxy ' ,carbonylV5-O-methyl-L-glutam-l-yl-2- fmethoxycarbonylVL-phenylalaninate To a dry 100 mL 3 -necked flask equipped with a thermometer, a magnetic stirrer, and a reflux condenser were added 1.50 g of methyl N-((benzyloxy)carbonyl)-5-O-methyl-L- glutam-l-yl-2-iodo-L-phenylalaninate, 0.149 g ofPd(Ph3P)4 (Aldrich), and 35 mL of 1 :1 MeΟH:THF under a nitrogen flush. The solution was deoxygenated by bubbling nitrogen through for 10 min and was then saturated with CO by bubbling CO through for 10 min. The solution changed color from yellow to bright orange during this period. The reaction vessel was put under CO balloon pressure by attaching a CO filled balloon to the condenser via a gas inlet adapter. The reaction mixture was heated to 60°C with stirring. After 3h, analysis by tic (Siθ2, 1:1 hexane:EtOAc) indicated no remaining starting material, a new component at Rf=0.40, and two minor components at Rf=0.90-0.95. The solution was cooled to RT and concentrated in vacuo to give a yellow solid. This material was purified by flash chromatography on 85 g of silica gel (1:1 hexane.EtOAc) to afford 1.23 g (93%) of methyl N-((benzyloxy)carbonyl)-5-O- methyl-L-glutam-l-yl-2-(methoxycarbonyl)-L-phenylalaninate as a white crystalline solid, m.p. 128-130°C. Example 23: Methyl 5-0-methyl-L-glutam-l-yl-2-fmethoxycarbonyl'.-L-phenylalaninate According to example 10, 1.10 g of methyl N-((benzyloxy)carbonyl)-5-O-methyl-L- glutam-l-yl-2-(methoxycarbonyl)-L-phenylalaninate was hydrogenolyzed in the presence of 0.214 g of 10% Pd(C) and 0.18 mL of acetyl chloride to afford 0.86 g (97%) of methyl 5-0-methyl-L-glutam-l-yl-2-(methoxycarbonyl)-L-phenylalaninate*HCl as a light yellow foam, m.p. 77-80°C. Example 24: Methyl N-fN-f 4-f f ( 1 ,2-dihvdro-3-methyl- 1 -oxo-benzof fiquinazolin-g-yπmethyπ-aminoV 2-fluorobenzoylV5-0-methyl-L-glutam-l-yl ' .-2-fmethoxycarbonyπ-L-phenylalaninate According to example 11, 0.50 g of 4-(((l,2-dihydro-3-methyl-l- oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoic acid was coupled with 0.55 g of methyl 5-O-methyl-L-glutam- 1 -yl-2-(methoxycarbonyl)-L-phenylalaninate*HCl using 0.17 mL of isobutyl chloroformate and 0.14 mL (2x) of N-methylmorpholine. The crude product was purified by flash chromatography on 250 g of silica gel (97:3—>95:5 CH2Cl2:MeOH) to afford 0.29 g (30%) of methyl N-(N-(4-((( 1 ,2-dihydro-3 -methyl- 1 -oxo-benzo(f)quinazolin-9-yl)methyl)amino)- 2-fluorobenzoyl)-5-O-methyl-L-glutam- 1 -yl)-2-(methoxycarbonyl)-L-phenylalaninate as a white powder, m.p. 180°C (dec). JH-ΝMR: (300 MHz, DMSO-d6) δ 12.52 (s, IH, benzoquinazoline ΝH), 9.86 (s, IH, aromatic CH), 8.52 (d, IH, J=7.6, amide ΝH), 8.23 (d, IH, J=8.8, aromatic CH), 8.03 (d, IH, J=8.4, aromatic CH), 7.80 (d, IH, J=7.2, aromatic CH), 7.59 (m, 2H, aromatic CH, 4-amino-benzoyl ΝH), 7.50 (t, IH, J=7.6, aromatic CH), 7.44-7.21 (m, 5H, aromatic CH, amide ΝH), 6.53 (d, IH, J=8.0, aromatic CH), 6.40 (d, IH, J=14.5, aromatic CH), 4.58 (m, 3H, benzoquinazoline 9-CH 2 , methine), 4.45 (m, IH, methine), 3.82 (s, 3H, ArCO2CH3), 3.57 (s, 3H, CO2CH3), 3.55 (s, 3H, CO2CH3), 3.48 (m, IH, phe Ar-CH2), 3.10 (m, IH, phe Ar-CH2), 2.45 (s, 3H, CH3), 2.27 (t, 2H, J=7.5, glu 4-CH2), 2.01-1.73 (m, 2H, glu 3-CH2). Example 25: N-fN-f4-fffl.2-Dihydro-3-methyl-l-oxobenzoff)quinazolin- 9-vPmethyl'-aminoV2-fluorobenzoyl ' .-L-glutam-l-yl ' ,-2-carboxy-L-phenylalanine A 50 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 0.11 g of methyl N-(N-(4-((( 1 ,2-dihydro-3-methyl- 1 -oxo-benzo(f)quinazolin-9-yl)methyl)amino)- 2-fluorobenzoyl)-5-O-methyl-L-glutam-l-yl)-2-(methoxycarbonyl)-L-phenylalaninate and 15 mL of 1:1 THF:H 2 O. The solution was treated with 62 mg of LiOH (Fisher Scientific Co., Fair Lawn, ΝJ) and stirred at RT under nitrogen. After 18h, analysis by HPLC (C18, 70:30:0.1 H 2 O:MeCΝ:TFA) showed no remaining starting material (k'=4.89), a new component at k'=2.29 (92%), and three minor components between k"=0.72 and 1.13. The solution was concentrated in vacuo to 7 mL and subjected to semipreparative HPLC (C18, 80:20:0.1 _.70:30:0.1 H 2 O:MeCN:TFA over 25 min). Pure fractions (as determined by analytical HPLC) were combined and concentrated in vacuo to dryness. The residue wasdissolved in 20 mL of0.17 M aqueous LiOH, filtered, and acidified to pH=3.5 by addition of IN aqueous HCl. A white solid resulted which was separated by centrifugation and washed with four cycles of aqueous suspension-centrifugation-decantation. The product was suspended in 10 mL of water, frozen, and lyophilized to afford 60 mg (56%) of N-(N-(4-(((l,2-dihydro-3-methyl- 1 -oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam- 1 -yl)- 2-carboxy-L-phenylalanine as a fluffy white solid, m.p. 245°C (dec). HPLC: one peak on C18, k'=1.86, 70:30:0.1 H 2 O:MeCΝ:TFA, flow rate=l mL/min. *H-NMR: (300 MHz, DMSO-d6) δ 13.00-11.70 (br s, COOH), 12.54 (s, IH, benzoquinazopline NH), 9.84 (s, IH, aromatic CH), 8.35 (d, IH, J=7.9, amide NH), 8.22 (d, IH, J=9.1, aromatic CH), 8.01 (d, IH, J=8.2, aromatic CH), 7.78 (d, IH, J=7.8, aromatic CH), 7.60 (m, 2H, aromatic CH, 4-aminobenzoyl NH), 7.50 (t, IH, J=7.8, aromatic CH), 7.45-7.18 (m, 5H, aromatic CH, amide NH), 6.53 (d, IH, J=8.5, aromatic CH), 6.39 (d, IH, J=14.7, aromatic CH), 4.67-4.46 (m, 3H, benzoquinazoline 9-CH , methine), 4.41 (m, IH, methine), 3.55 (dd; IH; J=4.0, 12.3; phe Ar-CH2), 3.06 (dd; IH, J=10.2, 12.9; phe Ar-CH 2 ), 2.42 (s, 3H, CH3), 2.16 (t, 2H, J=7.3, glu 4-CH2), 1.98-1.64 (m, 2H, glu 3-CH2). Elemental Analysis: Calcd. for C36H32N5O9F H2O (MW 715.69): C, 60.42; H, 4.79; N, 9.79. Found: C, 60.49; H, 4.83; N, 9.76. Mass Spectrum: (Ion Spray) 698 (M+H) + , 489, 361. UN Spectrum: (pH7 Buffer) λ max (ε) 265.2 (43700), 298.4 (25600), 347.9 (5320). lmin(e) 241.5 (20300), 284.9 (24600), 340.0 (3140). Example 26: Methyl N-f4-fff2.4-diamino-6-pteridinyBmethyπmethylamino enzoviy 5-O-methyl-L-glutam- 1 -yl-2-f methoxycarbonvD-L-phenylalaninate According to example 13, 0.29 g of 4-(N-(2,4-diaπ_ino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was reacted with 0.32 g of methyl 5-O-methyl-L-glutam-l-yl-2-(methoxycarbonyl)-L-phenylalaninate*HCl using 0.35 mL of DECP (Aldrich) and 0.58 mL of Et3Ν. The crude product was purified by flash chromatography on 100 g of silica gel (7:7:1 CH2C_2:acetone:MeOH) to afford 0.27 (51%) of methyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-methyl- L-glutam-l-yl-2-(methoxycarbonyl)-L-phenylalaninate as a yellow powder, m.p. 125-130°C. Example 27 : N-f 4-f f f 2.4-Diamino-6-pteridinyl ' .methyl'.methylamino.benzoyl ' . -L-glutam- 1 -yl-2-carboxy-L-phenylalanine To a 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 0.12 g of methyl N-(4-(((2,4-di_uτ_ino-6-pteridinyl)methyl)methylamino)benzoyl)- 5-0-methyl-L-glutam-l-yl-2-(methoxycarbonyl)-L-phenylalaninate and 10 mL of 1 : 1 H 2 O:THF. The mixture was treated with 29 mg of LiOH H 2 O (J.T. Baker Chemical Co., Phillipsburg, ΝJ), and was stirred at RT under nitrogen. After 48h, analysis by HPLC (C18, 75:25:0.1 H2θ:MeCΝ:TFA) indicated a major new component at k'=1.70 (ca. 90%) and three minor components at k'=0.60, 1.03, and 2.58. The solution was neutralized by dropwise addition of IN aqueous HCl and concentrated in vacuo to dryness. The residue was dissolved in 10 mL of MeOH, filtered, and diluted with 20 mL of water to precipitate the product. The MeOH was removed by rotary evaporation and the remaining suspension was frozen and lyophilized to afford 65 mg (45%) of -^-( 4_ ((( 2 , 4 -dian_dnc^6-pteridinyl)methyl)methyl_ιmino)benzoyl)-L-glutam-l-yl- 2-carboxy-L-phenylalanine as a yellow powder, m.p. 160°C (dec). HPLC: two peaks on C18; k'=2.49 (98%), k*=5.65 (2%); 78:22:0.1 H 2 O:MeCN:TFA; flow rate=l mLΛnin. 1 H*-NMR: (300 MHz, DMSO-d6) δ 13.10-11.90 (br5, COOH), 8.64 (s, IH, pteridinyl 7-CH), 8.62 (br s, IH, NH 2 ), 8.18 (d, IH, J=8.0, amide NH), 7.92 (d, IH, J=8.0, amide NH), 7.80-7.20 (br m, 2H, NH2), 7.77 (d, IH, J=7.3, aromatic CH), 7.70 (d, 2H, J=8.9, aromatic CH), 7.25 (m, 3H, aromatic CH), 6.81 (d, 2H, J=9.0, aromatic CH), 4.84 (s, 2H, pteridinyl-CH ), 4.49 (m, IH, methine), 4.39 (m, IH, methine), 3.51 (m, IH, phe Ar-CH 2 ), 3.24 (s, 3H, N-CH3), 3.08 (m, IH, phe Ar-CH2), 2.19 (t, 2H, J=7.6, glu 4-CH2), 1.97-1.72 (m, 2H, glu 3-CH2). Elemental Analysis: Calcd. for C30H3iN9Og-2.7H2O l.2TFA (MW 831.10): C, 46.82; H, 4.56; N, 15.17. Found: C, 46.76; H, 4.48; N, 15.15. Mass Spectrum: (Ion Spray) 646 (M+H) + , 410, 369, 185. UN Spectrum: (pH7 Buffer) λ max (ε) 258.8 (23400), 307.0 (24200), 372.8 (7540). lmin(e) 2 42.0 (16000), 273.2 (16200), 344.3 (5900). Example 28: Dimethyl N-ffbenzyloxy.carbonylV5-O-methyl-L-glutam-l-yl-L-aspartate According to example 9, 2.01 g of L-aspartic acid dimethylester-HCl (Sigma) was coupled with 3.00 g of N-((benzyloxy)carbonyl)-5-O-methyl-L-glutamic acid using 2.05 g of EDC (Aldrich) and 1.12 mL of N-methylmorpholine. This gave 3.93 g (88%) of dimethyl N-((benzyloxy)carbonyl)-5-0-methyl-L-glutam-l-yl-L-aspartate as a crystalline white solid, m.p. 86-88°C. Example 29: Dimethyl 5-O-methyl-L-glutam-l-yl-L-aspartate According to example 10, 2.00 g of dimethyl N-((benzyloxy)carbonyl)-5-O-methyl-L- glutam-1-yl-L-aspartate was hydrogenolyzed in the presence of 0.45 g of 10% Pd(C) and 0.39 mL of acetyl chloride to afford 1.56 g (100%) of dimethyl 5-Ο-methyl-L- glutam-l-yl-L-aspartate-HCl as a light yellow foam. JH-ΝMR: (200 MHz, DMSΟ-d6) δ 9.14 (d, IH, J=7.4, ΝH), 8.40 (br s, 3H, ΝH3+), 4.72 (m, IH, glu methine), 3.89 (m, IH, asp methine), 3.66 (s, 3H, CΟ 2 CH3), 3.65 (s, 3H, CΟ 2 CH3), 3.64 (s, 3H, CΟ 2 CH3), 2.85 (d, 2H, J=6.2, asp CH 2 ), 2.49 (m, 2H, glu 4-CH2), 2.04 (m, 2H, glu 3-CH 2 ). Example 30: Dimethyl N-(N-(4-((( 1.2-dihvdro-3-methyl-l-oxo-benzoff.quinazolin- 9-yπmethyl'.amino -2-fluorobenzovπ-5-O-methyl-L-glutam-l-ylVL-aspartate According to example 11, 0.50 g of 4-(((l,2-dihydro-3-methyl-l- oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoic acid was coupled with 0.45 g of dimethyl 5-O-methyl-L-glutam-l-yl-L-aspartateΗCl using 0.17 mL of isobutyl chloroformate and 0.14 mL (2x) of N-methyl morpholine. The crude product was purified by flash chromatography on 250 g of silica gel (95:5 CH2Cl2:MeOH) to afford 0.33 g (38%) of dimethyl N-(N-(4-((( 1 ,2-dihydro-3-methyl- 1 -oxo-benzo(f)quinazolin-9-yl)methyl)amino)-2- fluorobenzoyl)-5-O-methyl-L-glutam-l-yl)-L-aspartate as a white powder, m.p. 198-200°C. 1 H = ΝMR: (200 MHz, DMSO-d6) δ 12.54 (s, IH, benzoquinazoline NH), 9.85 (s, IH, aromatic CH), 8.55 (d, IH, J=7.7, amide NH), 8.23 (d, IH, J=8.8, aromatic CH), 8.02 (d, IH, J=8.4, aromatic CH), 7.70-7.45 (m, 4H, aromatic CH, amide NH), 7.33 (m, IH, 4-amino-benzoyl NH), 6.55 (dd; IH; J=8.8, 1.9; aromatic CH), 6.42 (dd; IH; J=15.3, 1.7; aromatic CH), 4.72-4.41 (m, 4H, benzoquinazoline 9-CH 2 , glu and asp methines), 3.62 (s, 3H, CO2CH3), 3.60 (s, 3H, CO2CH3), 3.55 (s, 3H, CO2CH3), 2.91-2.66 (m, 2H, asp CH2), 2.42 (s, 3H, CH3), 2.34 (t, 2H, J=7.7, glu 4-CH2), 2.13-1.80 (m, 2H, glu 3-CH2). Example 31: N-fN-f 4-ffl.2-Dihvdro-3 -methyl- 1-oxobenzoff.quinazolin- 9-yπmethyOamino V2-fluorobenzoylVL-glutam- 1 -yl-L-aspartic acid According to example 12, 0.12 g of dimethyl N-(N-(4-(((l,2-dihydro-3-methyl-l-oxo- benzo(f)quinazolin-9-yl) methyl)amino)-2-fluorobenzoyl)-5-O-methyl-L-glutam- 1 -yl)- L-aspartate was saponified in 8 mL of 1 : 1 THF-H2O using 45 mg of LiOHH2θ (Baker). Analysis of the crude product by reverse phase HPLC (C18, 80:20:0.1 H2θ:MeCΝ:TFA) indicated a major component at k-1.73 (90%) and a minor component at k'=1.28. The material was purified by semi-preparative HPLC using gradient elution (C18, 80:20:0.1 →75:25:0.1 H2θ:MeCN:TFA over 20 min ). Pure fractions (as determined by analytical HPLC) were combined and concentrated in vacuo. The residue was mixed with 20 mL of water and treated with enough LiOH H2θ to dissolve the solid. The solution was filtered and acidified to pH=3.0 by addition of IN HCl. A white solid precipitated which was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 57 mg (48%) of N-(N-(4-(( 1 ,2-dihydro-3-methyl- 1 -oxobenzo(f)quin__zolin-9-yl)methyl)amino)- 2-fluorobenzoyl)-L-glutam-l-yl-L-aspartic acid as an off-white solid, m.p. 185°C (dec). HPLC: one peak on C18, k'=1.42, 75:25:0.1 H2θ:MeCN:TFA, flow rate=l mL/min. *H-NMR: (300 MHz, DMSO-d6) δ 12.85-12.00 (br s, COOH), 12.53 (s, IH, benzoquinazoline NH), 9.84 (s, IH, aromatic CH), 8.36 (d, IH, J=7.9, amide NH), 8.21 (d, IH, J=8.8, aromatic CH), 8.01 (d, IH, J=8.3, aromatic CH), 7.67-7.46 (m, 4H, aromatic CH, amide NH), 7.32 (m, IH, 4-aminobenzoyl NH), 6.53 (d, IH, J=8.5, aromatic CH), 6.40 (d, IH, J=14.8, aromatic CH), 4.64-4.44 (m, 4H, benzoquinazoline 9-CH 2 , glu methine, asp methine), 2.75-2.53 (m, 2H, asp CH 2 ), 2.42 (s, 3H, CH3), 2.25 (t, 2H, J=7.3, glu 4-CH2), 1.98 (m, IH, glu 3-CH 2 ), 1.83 (m, IH, glu 3-CH 2 ). Elemental Analysis: Calcd. for C30H28N5O9F I.8H2O (MW 654.01): C, 55.10; H, 4.87; N, 10.71. Found: C, 55.04; H, 4.82; N, 10.61. Mass Spectrum: (Ion Spray) 622 (M+H) + , 316, 213, 156. UN Spectrum: (pH7 Buffer) λ max (ε) 264.8 (46200), 298.4 (28500), 347.8 (6180). lmin(e) 237.9 (19500), 285.7 (27900), 340.0 (3820). Example 32: Dimethyl N-f4-fff2.4-diamino-6-pteridinyπmethvDmethylamino benzoylV5-O-methyl-L- glutam- 1 -yl VL-aspartate According to example 13, 0.200 g of4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.18 g of dimethyl 5-O-methyl-L-glutam-l-yl-L-aspartate-HCl using 0.24 mL of DECP (Aldrich) and 0.41 mL of Et3N. The crude product was purified by flash chromatography on 100 g of silica gel (7:7:1 CH2C_2:acetone:MeOH) to afford 0.17 g (53%) of dimethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)- 5-O-methyl-L- glutam-l-yl)-L-aspartate as a yellow powder, m.p. 130-135°C. Example 33 : N-(4-ff f2.4-Diamino-6-pteridinv0methyl)methylamino ?enzoyl'.-L-glutam- 1-yl-L-aspartic acid fProcedure A) For Procedure B to prepare this compound, see Example 125. A 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.16 g of dimethyl Ν-(4-(((2,4-diamino- 6-pteridinyl)methyl)methylamino)benzoyl)-5-O-methyl-L- gluta - 1 -yl)-L-aspartate and 10 mL of 1 : 1 THF:H2θ. The solution was treated with 44 mg of LiOH H2O (Baker) and stirred at RT under N2. After 3h, tic (Siθ2, 7:7:1 CH2Cl2:acetone:MeOH) indicated no remaining starting material at Rf=0.30 and a single spot at R =0.0. Analysis by reverse phase HPLC (C18, 85:15:0.1 H2θ:MeCN:TFA) indicated a major component at k'=2.65 (90%) and a minor component at k'=1.94. The solution was neutralized by addition of IN HCl and concentrated in vacuo to dryness. The crude product was purified by semi-preparative HPLC (C18, 87:13:0.1 H2θ:MeCN:TFA). Pure fractions (as determined by analytical HPLC) were combined and concentrated in vacuo to dryness. The residue was dissolved in 20 mL of H2O and lyophilized to afford 65 mg (32%) of N-(4-(((2,4-di-_mino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl- L-aspartic acid as a yellow-orange powder, m.p. 165-180°C (dec). HPLC: one peak on C18, k'=2.45, 85:15:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. JH-NMR: (300 MHz, DMSO-d6) δ 13.00-11.85 (br s, COOH), 9.17 (br s, IH, NH2), 8.96 (br s, IH, NH2), 8.71 (s, IH, pteridinyl 7-CH), 8.60-8.30 (br s, IH, NH2), 8.21 (d, IH, J=8.0, amide NH), 8.06 (d, IH, J=7.8, amide NH), 7.85-7.43 (br s, IH, NH 2 ), 7.75 (d, 2H, J=8.7, aromatic CH), 6.82 (d, 2H, J=8.8, aromatic CH), 4.87 (s, 2H, pteridinyl-CH2), 4.59-4.38 (m, 2H, 2 methines), 3.25 (s, 3H, N-CH3), 2.75-2.53 (m, 2H, asp CH2), 2.28 (t, 2H, J=7.5, glu 4-CH2), 2.01 (m, IH, glu 3-CH2), 1.88 (m, IH, glu 3-CH2). Elemental Analysis: Calcd. for C24H27N9O8 1.9H2O 1.6TFA (MW 786.20): C, 41.55; H, 4.15; N, 16.03. Found: C, 41.53; H, 4.19; N, 16.00. Mass Spectrum: (Ion Spray) 570 (M+H) + , 223. UN Spectrum: (pH7 Buffer) λmax (ε) 258.7 (24400), 307.3 (26800), 372.9 (8080). lmin(e) 240.0 (14800), 272.9 (17000), 345.0 (6420). Example 34: Diethyl N-ffbenzyloxyteari onylV5-0-ethyl-L-glutaπ--l-yl-L-glutamate According to example 9, 3.17 g of L-glutamic acid diethyl ester HCl (Sigma) was coupled with 4.09 g of N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutamic acid using 2.66 g of EDC (Aldrich) and 1.45 mL of N-methylmorpholine. This gave 4.80 g (74%) of diethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-l-yl-L-glutamate as a white crystalline solid, m.p. 104°C. Example 35: Diethyl 5-O-ethyl-L-glutam-l-yl-L-glutamate To a 500 mL Parr bottle were added 0.75 of diethyl N-((benzyloxy)carbonyl)-5-O-ethyl- L-glutam-1-yl-L-glutamate, 0.14 g of 10% Pd(C) and 60 mL of EtOH under a Ν2 flush. The mixture was deoxygenated by bubbling N2 through for 7 min, treated with 1.52 mL of 1M ethereal HCl, and hydrogenated at 45 psi for 18h. The bottle was purged with N2, catalyst was removed by filtration through celite, and the filtrate was concentrated in vacuo to dryness to afford 0.60 (99%) of diethyl 5-O-ethyl-L-glutam-l-yl-L- glutamate HCl as a light yellow foam. *H-NMR: (300 MHz, DMSO-dό) δ 9.11 (d, IH, J=7.5, NH), 8.41 (br s, 3H, NH3 + ), 4.32 (m, IH, methine), 4.06 (m, 6H, ethyl CH2), 3.92 (m, IH, methine), 2.47 (m, 4H, glu 4-CH2), 2.12-1.79 (m, 4H, glu 3-CH2), 1.19 (t, 9H, J=7.1, ethyl CH3). Example 36: Diethyl N-(N-(4- ((( 1.2-dihvdro-3-methyl- 1 -oxobenzof f)quinazolin-9-yl'.methyl.amino ' .-2- fluorobenzoviy 5-O-ethyl-L-glutam- 1 -ylVL-glutamate According to example 11, 0.57 g of 4-(((l, 2-dihydro-3 -methyl- l-oxobenzo(f)quinazolin- 9-yl)methyl)amino)-2-fluorobenzoic acid was coupled with 0.60 g of diethyl 5-O-ethyl- L-glutam-1-yl-L-glutamate HCl using 0.20 mL of isobutyl chloroformate and 0.17 mL (2x) of N-methylmorpholine. The crude product was purified by flash chromatography on 350 g of silica gel (EtOAc→98:2 EtOAc.MeOH) to afford 0.27 g (25%) of diethyl N-(N-(4-((( 1 ,2-dihydro-3 -methyl- 1 -oxobenzo(f)quinazolin-9- yl)methyl)amino)-2-fluorobenzoyl)-5-O-ethyl-L-glutam-l-yl)-L-glutamate as a white powder, m.p. 185-187°C. Example 37 : N-fN- 4-f f f 1.2-Dihvdro-3-methyl- 1 -oxobenzof Oquinazolin- 9-yl ' .methyl ' . amino 2-fluorobenzovπ-L-glutam- 1 -ylVL-glutamic acid A 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.217 g of diethyl N-(N-(4-(((l,2-dihydro-3-methyl- l-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-ethyl-L-glutam- l-yl)-L-glutamate, 40 mL of 0.2 Ν aqueous ΝaOH, and 15 mL of EtOH. The mixture was stirred at RT under Ν2. The solid starting material slowly dissolved to give a clear solution. After 2h, tic (Siθ2, EtOAc) indicated no remaining starting material at Rf=0.15 and a new spot at Rf=0.0. The solution was acidified to pH=3.00 by addition of IN HCl. A white solid precipitated which was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 0.17 g (84%) of N-(N-(4-(((l,2-dihydro-3-methyl-l-oxobenzo(f)quinazolin- 9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-l-yl)-L-glutamic acid as a white powder, m.p. 180°C (dec). The product was determined to be pure by HPLC. HPLC: one peak on C18, k'=1.66, 75:25:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. 1 Ϊ£NMR: (300 MHz, DMSO-dό) δ 12.65-11.90 (br s, COOH), 12.53 (s, IH, benzoquinazoline NH), 9.84 (s, IH, aromatic CH), 8.29 (d, IH, J=7.5, amide NH), 8.22 (d, IH, J=8.9, aromatic CH), 8.01 (d, IH, J=8.0, aromatic CH), 7.60 (m, 3H, aromatic CH, amide NH), 7.51 (t, IH, J=9.0, aromatic CH), 7.33 (m, IH, 4-aminobenzoyl NH), 6.54 (dd; IH; J=8.5, 1.9; aromatic CH), 6.41 (dd; IH; J=13.9, 1.4; aromatic CH), 4.57 (d, 2H, J=5.2, benzoquinazoline 9-CH 2 ), 4.49 (m, IH, methine), 4.22 (m, IH, methine), 2.43 (s, 3H, CH3), 2.28 (m, 4H, glu 4-CH2), 2.10-1.70 (m, 4H, glu 3-CH2). Elemental Analysis: Calcd. for C31H30N5O9F 2H2O (MW 671.64): C, 55.44; H, 5.10; N, 10.43. Found: C, 55.38; H, 5.12; N, 10.26. Mass Spectrum: (FAB) 636 (M+H) + , 613, 581, 549. UN Spectrum: (pH7 Buffer) λ m ax (ε) 264.9 (45100), 296.1 (28000), 331.4 (7280), 348.1 (5990). -m in(e) 285.6 (27200), 329.3 (7040), 339.8 (3700). l s h(e) 271.5 (41900). Example 38: Diethyl N-f4-fff2.4-diamino-6-pteridinyl ' .methyl ' .methylamino ' .benzoylV5-O-ethyl- L-glutam- 1 -yl-L-glutamate According to example 13, 0.142 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.15 g of diethyl 5-O-ethyl-L-glutam-l-yl-L-glutamate-HCl using 0.18 mL of DECP (Aldrich) and 0.31 mL of Et3Ν. The crude product was purified by flash chromatography on 120 g of silica gel (7:7:1 CH 2 Cl 2 :acetone:MeOH) to afford 0.14 g (56%) of diethyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)- 5-O-ethyl-L-glutam-l-yl-L-glutamate as a yellow powder, m.p. 105-109°C. Example 39: N-f4-fff2.4-Di_ιπ_ino-6-pteridinyl)methyDmethylan_rino)benzoyD- L-glutam-1-yl-L-glutamic acid fProcedure A) For Procedure B to prepare this compound, see Example 129. According to example 33, 0.125 g of diethyl N-(4-(((2,4-diamino- 6-pteridinyl)methyl)methylamino)benzoyl)-5-0-ethyl-L-glutam- 1 -yl-L-glutamate was saponified in 10 mL of 1:1 THF:H2θ using 23 mg of LiOH (Fisher). After stirring at RT under Ν2 for 18 h, the solution was acidified to pH=2.0 by addition of IN HCl and was then concentrated in vacuo to dryness. Analysis of the residue by HPLC (C18, 85:15:0.1 H2θ:MeCN:TFA) indicated a major component at k-2.89 (90%) and two minor components at k'=2.46 and 1.17. The crude product was purified by semi-preparative HPLC (C18, 87:13:0.5 H2θ:MeCN:TFA) and lyophilized to afford 37 mg (24%) of *^-( 4 -((( 2 , 4 -dian_dno-6-pteridinyl)methyl)methyl__mino)benzoyl)-L-glutam-l-yl- L-glutamic acid as a yellow powder, m.p. 178°C. HPLC: one peak on C18, k'=2.90, 85:15:0.1 H2θ:MeCN:TFA, flow rate=l mL/min. *H-NMR: (300 MHz, DMSO-d6) δ 13.20-12.00 (br s, COOH), 9.30 (br s, IH, NH2), 9.10 (br s, IH, NH2), 8.74 (s, IH, pteridinyl 7-CH), 8.72-8.42 (br s, IH, NH2), 8.20 (d, IH, J=7.5, amide NH), 8.07 (d, IH, J=7.5, amide NH), 7.76 (d, 2H, J=8.8, aromatic CH), 7.61-7.40 (br s, IH, NH2), 6.83 (d, 2H, J=8.8, aromatic CH), 4.90 (s,2H, pteridinyl-CH2), 4.43 (m, IH, methine), 4.20 (m, IH, methine), 3.27 (s, 3H, N-CH3), ,2.30 (m, 4H, glu 4-CH2), 2.08-1.77 (m, 4H, glu 3-CH2). Elemental Analysis: Calcd. for C25H29 9O8 2.3H2O 1.7TFA (MW 818.84): C, 41.66; H, 4.35; N, 15.40. Found: C, 41.61; H, 4.25; N, 15.44. Mass Spectrum: (FAB) 584 (M+H) + , 309, 275, 176, 155, 119. UN Spectrum: (pH7 Buffer) λ max (ε) 220.9 (20500), 258.5 (21500), 306.7 (23200), 372.5 (7180). lmin(e) 240.1 (13400), 272.5 (15100), 344.8 (5730). Example 40: 3-Cvano-L-tyrosine A solution of 5.70 g of 3-amino-L-tyrosine (Aldrich) in 35 mL of 2.15 Ν aqueous HCl was cooled to 0°C and treated with a solution of 1.41 g of ΝaΝθ2 (Aldrich) in 5 mL of water followed by 2.97 g of a2CO3. After stirring at 0°C for 7 min, the solution was slowly added (via an addition funnel) to a stirred mixture of 3.58 g of CuCN (Aldrich), 5.88 g of NaCN (Aldrich) and 5.72 g of Na2CO3 in 40 mL of water maintained at 75°C in a 250 mL 3-necked flask equipped with a condenser. During the addition, vigorous evolution of nitrogen occurred and the reaction mixture changed color to dark red-orange. The reaction mixture was stirred at 75°C for 2h, heated to 95°C, and stirred for an additional lh. After cooling to RT, the reaction mixture was acidified to pH=2.0 by addition of concentrated HCl via the addition funnel. The evolved gas was scrubbed through a 20% aqueous NaOH trap followed by a 5% NaOCl (Chlorox) trap. The mixture was filtered and the pH of the filtrate was adjusted to 6.0 with concentrated NH4OH. The brown solution was mixed with 300 mL of water and stirred with 300 g of DOWEX 50WX-8 ion exchange resin (acid form). The resin was filtered, washed with IL of water, mixed with 500 mL of water, and the mixture was basified to pH 11 by addition of concentrated NH4OH. The mixture was filtered and the filtrate concentrated in vacuo to dryness. The dark brown residue was suspended in 20 mL of water and treated with enough IN aqueous NaOH to dissolve the solid. The pH was then adjusted to 6.0 by addition of IN aqueous HCl. A precipitate formed which was collected by filtration and dried in vacuo to afford 1.45 g (35%) of 3-cyano-L-tyrosine as a chocolate brown solid, m.p. >250°C. JH-NMR: (200 MHz, DMSO-dό) δ 9.60-7.20 (br s, NH2), 7.39 (s, IH, aromatic CH), 7.30 (m, IH, aromatic CH), 6.98 (m, IH, aromatic CH), 3.52 (m, IH, methine), 2.92 (m, 2H, ArCH2). Example 41: Methyl S-fmethoxycarbonylVL-tyrosinate A 250 mL 3-necked flask equipped with a reflux condenser and a magnetic stirrer was charged with 1.45 g of 3-cyano-L-tyrosine, 50 mL of concentrated HCl, and 50 mL of water. The solution was heated to reflux with stirring. After 2 days, analysis of the reaction mixture by HPLC (C18, 90:10:0.1 H2θ:MeCN:TFA) indicated a major component at k'=2.23 (75%) and a minor component at k'=l .59 (25%). After 4 days HPLC indicated only the k'=2.23 material. The reaction mixture was cooled to RT, filtered, and the filtrate was concentrated in vacuo to dryness to give 1.2 g of 3-carboxy- L-tyrosine as a dark brown solid. IR analysis (nujol) indicated loss of the nitrile stretch of 3-cyano-L-tyrosine, at 2223 cm"*-. The crude solid was added to 60 mL of anhydrous MeOH in a 250 mL 3-necked flask equipped with a condenser and a magnetic stirrer. The mixture was acidified by bubbling HCl gas through for 5 min and heated to reflux with stirring. After 6h, the reaction mixture was cooled to RT and neutralized with solid NaHCO3. The mixture was concentrated in vacuo to dryness, mixed with 100 mL of water, and extracted with CH2CI2 (4x50 mL). The combined extracts were washed with 5% aqueous NaHCO3 (2 x 50 mL), dried over anhydrous Na2Sθ4, and concentrated in vacuo to 10 mL. The solution was diluted with 35 mL of ether and treated with 5.5 mL of 1M ethereal HCl. A solid precipitated which was collected by filtration, washed with ether, and dried in vacuo to afford 0.90 (50%) of methyl 3- (methoxycarbonyl)-L-tyrosinate HCl as an off-white powder, m.p. 211-213°C. iH-NMR: (300 MHz, DMSO-d6) δ 10.45 (s, IH, OH), 8.59 (br s, 3H, NH3 + ), 7.66 (s, IH, aromatic CH), 7.38 (dd; IH; J=7.9, 1.1, aromatic CH), 6.97 (d, IH, J=7.9, aromatic CH), 4.26 (m, IH, methine), 3.89 (s, 3H, CO2CH3), 3.68 (s, 3H, CO2CH3), 3.09 (m, 2H, A1-CH2). Example 42: Methyl N-ffbenzyloxytearbonylV5-0-methyl-L-glutam-l-yl-3- fmethoxycarbonylVL-tyrosinate According to example 9, 0.80 g of methyl 3-(methoxycarbonyl)-L-tyrosinate*HCl was coupled with 0.82 g of N-((benzyloxy)carbonyl)-5-O-methyl-L-glutamic acid using 0.56 g of EDC (Aldrich) and 0.30 mL of N-methylmorpholine. This gave 1.24 g (85%) of methyl N-((benzyloxy)carbonyl)-5-0-methyl-L-glutam-l-yl-3-(methoxycarbonyl)-L- tyrosinate as a white solid, m.p. 142-144°C. Example 43 : Methyl 5-O-methyl-L-glutam- 1 -yl-3-f methoxycarbonylVL-tyrosinate According to example 10, 0.99 g of methyl N-((benzyloxy)carbonyl)-5-O-methyl-L- glutam-l-yl-3-(methoxycarbonyl)-L-tyrosinate was hydrogenolyzed using 0.19 g of 10% Pd(C) and 0.15 mL of acetyl chloride in 90 mL of 2:1 EtOH.EtOAc to afford 0.75 g (93%) of methyl 5-O-methyl-L-glutam-l-yl-3-(methoxycarbonyl)-L-tyrosinate*HCl as a light yellow foam, m.p. 95-97°C (dec). Example 44 : Methyl N-fN- 4f f 1.2-dihydro-3-methyl- 1 -oxobenzof f.quinazolin- 9-vπmethyπaminoV2-fluorobenzoylV5-O-methyl-L-glutam-l-yπ-3-fmethoxycarbonyl - L-tyrosinate According to example 11, 0.65 g of 4-(((l,2-dihydro-3-methyl-l-oxobenzo(f)quinazolin- 9-yl)methyl)amino)-2-fluorobenzoic acid was coupled with 0.75 g of methyl 5-O-methyl- L-glutam-l-yl-3-(methoxycarbonyl)-L-tyrosinate*HCl using 0.22 mL of isobutyl chloroformate and 0.19 mL (2x) of N-methylmorpholine. The crude product was purified by flash chromatography on 300 g of silica gel (99:l-» 94:6 EtOAcMeOH) to afford 0.53 g (40%) of methyl N-(N-(4(((l,2-dihydro-3-methyl-l-oxobenzo(f)quinazolin- 9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-methyl-L-glutam-l-yl)-3-(methoxycarbonyl)- L-tyrosinate as a white powder, m.p. 195-203°C (dec). ••H-ΝMR: (300 MHz, DMSO-dό) δ 12.52 (benzoquinazoline ΝH), 10.38 (s, IH, OH), 9.83 (s, IH, aromatic CH), 8.45 (d, IH, J=7.5, amide ΝH), 8.21 (d, IH, J=8.9, aromatic CH), 8.00 (d, IH, J=8.3, aromatic CH), 7.60 (m, 3H, aromatic CH, 4-aminobenzoyl ΝH), 7.48 (m, 2H, aromatic CH), 7.36 (m, 2H, aromatic CH, amide ΝH), 6.86 (d, IH, J=8.6, aromatic CH), 6.52 (dd; IH; J=8.5, 1.9; aromatic CH), 6.39 (dd; IH; J=13.9, 1.5; aromatic CH), 4.57 (d 2H, J=5.6, benzoquinazoline 9-CH2), 4.45 (m, 2H, methines), 3.84 (s, 3H, CO2CH3), 3.58 (s, 3H, CO2CH3), 3.52 (s, 3H, CO2CH3), 3.05-2.80 (m, 2H, ArCH2), 2.42 (s, 3H, CH3), 2.25 (t, 2H, J=7.8, glu 4-CH2), 2.00-1.77 (m, 2H, glu 3-CH2). Example 45: 3-Carboxy-N-fN-f4-fffl.2-dihvdro-3-methyl-l-oxobenzoff.quinazolin- 9-yl~.methvn__iT-ino V2-fluorobenzoyl VL-glutam- 1 -ylVL-tyrosine A solution of 0.15 g of methyl N-(N-(4(((l,2-dihydro-3-methyl-l-oxobenzo(f)quinazolin- 9-yl)methyl)amino)-2-fluorober_zoyl)-5-O-methyl-L-glutam- 1 -yl)-3 -(methoxycarbonyl)- L-tyrosinate in 20 mL of 0.2 Ν aqueous ΝaOH was stirred at RT under Ν2 for 18h. Analysis of the solution by HPLC (C18, 70:30:0.1 H 2 O:MeCN:TFA) indicated a major component at k-1.9 (90%) and two minor components at k'=1.0 and 2.6. The solution was acidified to pH 2.5 by addition of IN HCl. A white precipitate resulted which was separated by centrifugation and purified by semi-preparative HPLC (C18, 75:25:0.1 H2θ:MeCN:TFA). Pure fractions (as determined by analytical HPLC) were combined and concentrated in vacuo to dryness. The residue was dissolved in 20 mL of 0.15 N aqueous NaOH and the solution was acidified to pH=3.0 by addition of IN HCl. The resulting white precipitate was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 39 mg (26%) of 3-carboxy-N-(N-(4-((( 1 ,2-dihydro-3-methyl-l -oxobenzo(f)quinazolin- 9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-l-yl)-L-tyrosine as a white powder, m.p. 198-205°C (dec). HPLC: one peak on C18, k'=1.86, 70:30:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. -tH-NMR: (200 MHz, DMSO-d6) δ 13.00-11.00 (br m, COOH, benzoquinazoline NH), 9.86 (s, IH, aromatic CH), 8.28 (d, IH, J=7.5, amide NH), 8.24 (d, IH, J=8.9, aromatic CH), 8.03 (d, IH, J=8.4, aromatic CH), 7.70-7.42 (m, 5H, aromatic CH, 4-aminobenzoyl NH), 7.40-7.24 (m, 2H, aromatic CH, amide NH), 6.78 (d, IH, J=8.6, aromatic CH), 6.55 (dd; IH; J=8.5, 1.9; aromatic CH), 6.42 (dd; IH; J=13.9, 1.5; aromatic CH), 4.58 (d, 2H, J=5.8, benzoquinazoline 9-CH2), 4.55-4.31 (m, 2H, methines), 3.06-2.75 (m, 2H, ArCH2), 2.24 (s, 3H, CH3), 2.21 (t, 2H, J=7.6, glu 4-CH 2 ), 2.08-1.70 (m, 2H, glu 3-CH 2 ). Elemental Analysis: Calcd. for C36H32N5O10F 2.2 H 2 O (MW 753.31): C, 57.40; H, 4.87; N, 9.30. Found: C, 57.37; H, 4.97; N, 9.28. Mass Spectrum: (FAB) 714 (M+H) + , 613, 549, 461, 360, 309. UN Spectrum: (pH7 Buffer) lmax(e) 265.9 (44700), 300.2 (28200), 348.3 (5270). lmin(e) 246.3 (23400), 285.2 (25500), 340.8 (3630). Example 46: fert-Butyl 3-ftert-butoxycarbonyD-L-tyrosinate A 250 mL 3-necked flask equipped with a magnetic stirrer and a condenser was charged with 4.1 g of 3-cyano-L-tyrosine and 100 mL of 6Ν aqueous HCl. The mixture was heated to reflux with stirring. After 36h, HPLC (C18, 90:10:0.1 H 2 O:MeCN:TFA) indicated no remaining starting material at k'=1.33 and a major new component at k'=1.69. The reaction mixture was cooled to RT, filtered to remove solids, and concentrated in vacuo to dryness. The residue was dissolved in 100 mL of 8:2 H2θ:MeOH and the solution was passed through two C18 plugs (3.5 x 4.5 cm, EM separations LiChroprep RP-18) washing with 8:2 H 2 O:MeOH. The filtrate was concentrated in vacuo to dryness to afford 4.5 g of 3-caιboxytyrosine as a brown solid. A portion of this material (1.30 g) was dissolved in 40 mL of 1,4-dioxane with 2 mL of concentrated H2SO4 and the solution was added to 30 mL of isobutylene (condensed at -78°C) in a 300 mL pressure bottle equipped with a magnetic stirrer. The vessel was stoppered, warmed to RT and stirred. After 48h, the bottle was cooled to 0°C in an ice water bath, opened, and the mixture was treated with 20 g of NaHCO3. After allowing the isobutylene to evaporate, the mixture was suspended in 300 mL of water and extracted with CH2CI2 (4 x 100 mL). The combined CH2CI2 extracts were washed with 5% aqueous NaHCO3 (2 x 50 mL), dried over anhydrous MgSO4, and concentrated in vacuo to give a yellow-brown oil. Analysis by tic (Siθ2, 97:3 CH 2 Cl 2 :MeOH) indicated a major component at Rf=0.41 and three minor components at R =0.30, 0.25 and 0.0. The crude material was purified by flash chromatography on 75 g of silica gel (94:6 CH2Cl 2 :MeOH) to give 1.2 g of a light yellow oil. The oil was dissolved in 40 mL of ether and treated with 3.9 mL of 1M ethereal HCl. A precipitate formed which was collected by filtration and dried in vacuo to afford 1.14 g (53%) of tert-butyl 3-(tert-butoxycarbonyl)-L-tyrosinateΗCl as a fluffy white solid. An analytical sample (26 mg) was prepared by recrystallization from MeOH-ether, m.p. 205°C (dec). JH-NMR: (300 MHz, DMSO-d6) δ 10.69 (s, IH, OH), 8.39 (br s, 3H, NH3 + ), 7.57 (d, IH, J=2.2, aromatic CH), 7.43 (dd; IH; J=8.6, 2.2; aromatic CH), 6.96 (d, IH, J=8.4, aromatic CH), 4.12 (m, IH, methine), 3.11 (dd; IH; J=14.3, 5.3; ArCH2), 2.97 (dd; IH, J=14.1, 8.1; ArCH2), 1.57 (s, 9H, t-Bu), 1.36 (s, 9H, t-Bu). Example 47: tert-Butyl N-f fbenzyloxykarbonylV5-O-tert-butyl-L-glutam- 1 -yl-3-f tert- butoxycarbonylVL-tyrosinate According to example 9, 0.323 g of tert-butyl 3-(/ert-butoxycarbonyl)-L-tyrosinate*HCl was coupled with 0.292 g of Ν-benzyloxycarbonyl-L-glutamic acid γ-t-butyl ester (Sigma) using 0.174 g of EDC (Aldrich) and 0.095 mL of N-methylmorpholine. The crude product was purified by flash chromatography on 85 g of silica gel (75:25 hexane:EtOAc) to afford 0.49 g (89%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert- butyl-L-glutam-l-yl-3-(tert-butoxycarbonyl)-L-tyrosinate as a tacky white foam. JH-ΝMR: (300 MHz, DMSΟ-d6) δ 10.67 (s, IH, ΟH), 8.21 (d, IH, J=7.4, ΝH), 7.56 (s, IH, aromatic CH), 7.46-7.26 (m, 7H, aromatic CH, ΝH), 6.86 (d, IH, J=8.5, aromatic CH), 5.00 (m, 2H, PhCH2θ), 4.31 (m, IH, methine), 4.06 (m, IH, methine), 2.89 (d, 2H, J=6.9, ArCH2), 2.21 (t, 2H, J=7.7, glu 4-CH2), 1.92-1.64 (m, 2H, glu 3-CH2), 1-57 (s, 9H, t-Bu), 1.38 (s, 9H, t-Bu), 1.33 (s, 9H, t-Bu). Example 48: /ert-Butyl 5-0-tert-butyl-L-glutam-l-yl-3-ftert-butoxycarbonylVL- tyrosinate To a 300 mL Parr bottle were added 0.076 g of 10% Pd(C) and 20 mL of MeOH under a nitrogen flush. To this was added a solution of 0.49 g of tert-butyl N- ((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-l-yl-3-(tert-butoxycarbonyl)-L- tyrosinate in 50 mL of MeOH. The mixture was deoxygenated by bubbling nitrogen through for 7 min and was hydrogenated at 50 psi for 3h. The vessel was purged with nitrogen, catalyst removed by filtration through celite, and the filtrate concentrated in vacuo to give a thick oil. This material was purified by flash chromatography on 70 g of silica gel (98:2 CH 2 Cl 2 :MeOH) to afford 0.31 g (82%) of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3- (/ert-butoxycarbonyl)-L-tyrosinate as a thick transparent oil. A small sample of the corresponding HCl salt for -1H-NMR analysis was prepared by treating 5 mg of the product with excess IN ethereal HCl and concentrating to dryness. JH-NMR: (200 MHz, DMSO-dό) δ 10.68 (s, IH, OH), 8.99 (d, IH, J=7.1, NH), 8.33 (br s, 3H, NH3 + ), 7.60 (d, IH; J=2.0, aromatic CH), 7.47 (dd; IH; J=8.6, 2.2; aromatic CH), 6.92 (d, IH, J=8.4, aromatic CH), 4.38 (m, IH, methine), 3.87 (m, IH, methine), 2.92 (d, 2H, J=7.1, ArCH2), 2.34 (m, 2H, glu 4-CH2), 2.00 (m, 2H, glu 3-CH 2 ), 1.59 (5, 9H, t-Bu), 1.41 (s, 9H, t-Bu), 1.35 (s, 9H, t-Bu). Example 49: /ert-Butyl N-f4-fff2.4-diamino-6-pteridinyDmethvπmethylamino'.benzoyO- 5-O-tert-butyl-L-glutam- 1 -yl-3-f tert-butoxycarbonylVL-tyrosinate A dry 100 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 25 mL of anhydrous DMF, 0.26 mL of DECP (Aldrich) and 0.28 mL of Et3Ν. To this solution was added 0.217 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich). The solid starting material slowly dissolved to give a light yellow solution. After stirring for 3h under Ν2, the solution was treated with 0.29 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3-(tert- butoxycarbonyl)-L-tyrosinate in 5 mL of DMF. The solution was stirred for 18h at RT and was then concentrated in vacuo to dryness. The residue was dissolved in 85 mL of CHCI3. The resulting solution was washed with 1% aqueous NH4OH (3x60 mL), dried over anhydrous MgSO4, and concentrated to give a thick, yellow-orange oil. This material was purified by flash chromatography on 150 g of silica gel (7:7: 1 CH Cl2:acetone: MeOH) to afford 0.245 g (52%) of tert-butyl N-(4-(((2,4-diamino- 6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3-(tert- butoxycarbonyl)-L-tyrosinate as a light yellow powder, m.p. 140-143°C. iH-NMR: (300 MHz, DMSO-d6) δ 10.64 (s, IH, OH), 8.57 (s, IH, pteridinyl 7-CH), 8.15 (d, IH, J=7.4, amide NH), 7.99 (d, IH, J=7.9, amide NH), 7.71 (d, 2H, J=8.8, aromatic CH), 7.67 (br s, IH, NH2), 7.53 (d, IH, J=2.0, aromatic CH), 7.43 (br s, IH, NH2), 7.37 (dd; IH; J=8.6, 2.1; aromatic CH), 6.81 (m, 3H, aromatic CH), 6.59 (br s, 2H, NH2), 4.79 (s, 2H, pteridinyl-CH 2 ), 4.43 (m, IH, methine), 4.32 (m, IH, methine), 3.21 (s, 3H, N-CH3), 2.90 (d, 2H, J=7.1, ArCH2), 2.24 (t, 2H, J=7.9, glu 4-CH2), 2.03-1.78 (m, 2H, glu 3-CH2), 1.54 (5, 9H, t-Bu), 1.36 (s, 9H, t-Bu), 1.30 (s, 9H, t-Bu). Example 50: N-f4-fff2.4-Diamino-6-pteridinyl methyl ' )methylamino ' )benzoyl')- L-glutam- 1 -yl-3 -carboxy-L-tyrosine A 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.20 g of tert-Butyl Ν-(4-(((2,4-diamino- 6-pteridinyl)methyl)methylamino)benzoyl)-5-0- tert-butyl-L-glutam- 1 -yl-3-(tert- butoxycarbonyl)-L-tyrosinate and 20 mL of CH3NO2. The yellow solution was stirred at RT and acidified by bubbling gaseous HCl through for 10 min. Upon HCl treatment, the color rapidly changed from yellow to orange to lighter yellow and a solid began to precipitate. The mixture was stirred at RT under N 2 for 1.5 h and was then concentrated in vacuo to dryness. The resulting yellow solid was suspended in 15 mL of water and treated with sufficient IN aqueous NaOH to give complete solution. The solution was filtered and neutralized by addition of IN aqueous HCl. A yellow precipitate resulted which was separated by centrifugation, washed with four cycles of aqueous suspension- centrifugation-decantation, and lyophilized to afford 140 mg (88%) of N-(4-(((2,4-Di_-n__ino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl- 3-carboxy-L-tyrosine as a yellow-orange powder, m.p. 195°C (dec). HPLC: two peaks on C18; k'=3.16 (98%), k'=6.07 (2%); 80:20:0.1 H2θ:MeCΝ:TFA:flow rate=l mL/min. iH-NMR: (300 MHz, DMSO-d6) δ 13.15-11.80 (br s, COOH), 8.62 (s, IH, pteridinyl 7-CH), 8.19 (br s, IH, NH2), 8.00 (m, 3H, amide NH (2), NH2), 7.72 (d, 2H, J=8.7, aromatic CH), 7.61 (d, IH, J=2.1, aromatic CH), 7.22 (m, 3H, NH2(2), aromatic CH), 6.81 (d, 2H, J=8.7, aromatic CH), 6.66 (d, IH, J=7.8, aromatic CH), 4.81(s, 2H, pteridinyl-CH 2 ), 3.39 (m, 2H, methines), 3.21 (s, 3H, N-CH3), 2.94 (m, IH, ArCH 2 ), 2.86 (m, IH, ArCH2), 2.27 (t, 2H, J=7.9, glu 4-CH2), 2.05-1.72 (m, 2H, glu 3-CH2). Elemental Analysis: Calcd. for C30H31Ν9O9 I.8H2O (MW 694.06): C, 51.92; H, 5.02; N, 18.16. Found: C, 51.84; H, 5.0Z; N, 18.19. Mass Spectrum: (Ion Spray) 662 (M+H) + , 385, 306, 235, 157. UN Spectrum: (pH7 Buffer) λ ma χ (ε) 258.6 (19900), 306.3 (23500), 372.6 (5490). lmin(e) 245.4 (14800), 272.8 (12600), 347.0 (4040). Example 51: (2R. 3S. 5S.-Benzyl 3-f3-iodobenzylV2-oxo-2.3-diphenyl-4- mo holinecarboxylate A dry 500 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 3.0 g of benzyl (2R, 3S)-6-oxo-2,3-diphenyl-4-morpholinecarboxylate (Aldrich) and 180 mL of anhydrous THF. After gently warming the mixture to dissolve the solid starting material, the solution was cooled to -78°C and was treated with 8.13 mL of 1M sodium bis(trimethylsilyl)amide in THF (Aldrich) by slow addition via syringe through a rubber septum. The solution was stirred at -78°C under Ν2 for 40 min and was then treated with a solution of 2.41 g of 3-iodobenzyl bromide (Lancaster, Windham, NH 03087) in 5 mL of anhydrous THF. The reaction mixture was stirred at - 78°C for 2.5h, mixed with 100 mL of water and the resulting mixture was concentrated in vacuo to dryness. The residue was dissolved in 100 mL of EtOAc, washed with water (3x100 mL), dried over anhydrous Na2S04, and concentrated in vacuo to dryness to give a tan solid. Analysis by tic (Siθ2, 8:2 hexane:EtOAc) indicated no remaining starting material at R 0.35, a major new component at Rf=0.50 and some unreacted 3- iodobenzyl bromide at Rf=0.80. The crude product was purified by flash chromatography on 150 g of silica gel (8:2 hexane:EtOAc) to afford 2.8 g (60%) of (2R, 3S, 5<S)-benzyl 3-(3-iodobenzyl)-2-oxo-2,3-diphenyl-4-moφholinecarboxylate as a white crystalline solid, m.p. 167-168°C. Example 52: f2R. 3S. 5_SVBenzyl 3-f3-methoxycarbonvD-2-oxo-2-3-diphenyl-4- moφholinecarboxylate A dry 100 mL 3-necked flask equipped with a magnetic stirrer, a reflux condenser, and a thermometer was charged with 2.56 g of (2R, 3S, 5S)-benzyl 3-(3-iodobenzyl)-2-oxo- 5,6-diphenyl-4-moφholinecarboxylate and 50 mL of 1 : 1 THF:MeOH. The mixture was deoxygenated by bubbling N2 through for 10 min and was treated with 1.0 mL of Et3N followed by 0.24 g of Pd(Ph3P)4 (Aldrich). The mixture was saturated with CO and was heated to reflux (65°C) with stirring. A slight positive pressure of CO was maintained by attaching a CO gas inlet adapter equipped with a mineral oil bubbler to the condenser. After 4h at reflux, tic (Siθ2, 8:2 hexane.EtOAc) indicated no remaining starting material at Rf=0.50 and a new component at Rf=0.30. The reaction mixture was cooled to RT at which point a voluminous white solid precipitated. The suspension was mixed with 100 mL of CH2CI2 and the resulting solution was filtered through a silica gel plug (2.5x3 cm) and concentrated in vacuo to give a light yellow solid. This material was purified by flash chromatography on 150 g of silica gel (8:2 hexane:EtOAc) to afford 2.0 g (88%) of (2R, 3S, 5S)-benzyl 3-(3-methoxycarbonyl)-2-oxo-5,6-diphenyl-4- moφholinecarboxylate as an off-white crystalline solid, m.p. 180-182°C. *H-NMR: (200 MHz, DMSO-d6) δ 8.05-7.80 (m, 2H, aromatic CH), 7.72-6.97 (m, 14H, aromatic CH), 6.73 (m, IH, aromatic CH), 6.58 (m, 2H, aromatic CH), 6.25, 6.16 (d, J=2.2; d, J=2.6; IH total; 2 resonances for moφholine 2-H due to carbamate conformers), 5.36, 5.31 (d, J=2.6; d, J=2.6; IH total; 2 resonances for moφholine 3-H due to carbamate conformers), 5.16-4.88 (m, 3H, moφholine 5-H, PhCH2θ), 3.88, 3.85 (5, 5, 3H total, CO2CH3, conformers), 3.71-3.40 (m, 2H, ArCH2). Example 53: 3-Methoxycarbonyl-L-phenylalanine To a 500 mL Parr bottle were added 2.0 g of (2R, 3S, 5S)-benzyl 3-(3- methoxycarbonyl)-2-oxo-5,6-diphenyl-4-moφholinecarboxylate, 0.46 g of PdCl2 (Aldrich) and 60 mL of 1 : 1 THF MeOH. The mixture was deoxygenated by bubbling N2 through for 10 min and was then hydrogenolyzed at 45 psi for 18 h. The bottle was purged with N2 and the mixture filtered to remove catalyst. The filtrate was concentrated in vacuo to 5 mL and triturated with addition of 50 mL of ether. A white solid precipitated which was collected by vacuum filtration, washed with 30 mL of ether, and dried in vacuo to afford 0.88 g (91%) of 3-methoxycarbonyl-L-phenylalanineΗCl, m.p. >200 O C. H-NMR: (200 MHz, DMSO-dό) δ 8.50 (br s, 3H, NH3 + ), 7.89 (m, 2H, aromatic CH), 7.67-7.43 (m, 2H, aromatic CH), 4.21 (m, IH, methine), 3.87 (s, 3H, CO2CH3), 3.22 (d, 2H, J=6.5, ArCH2). Example 54: tert-Butyl 3-methoxycarbonyl-L-phenylalaninate A solution of 0.88 g of 3-methoxycarbonyl-L-phenylalanineΗCl, and 1.5 mL of cone H2SO4 in 20 mL of 1,4-dioxane was added to 25 mL of liquid isobutylene (condensed at -78°C) in a 300 mL pressure bottle equipped with a magnetic stirrer. The bottle was sealed and the mixture allowed to warm to RT with stirring. The initially heterogeneous mixture afforded a slightly cloudy solution after stirring for 18h at RT. After 48h the vessel was chilled in an ice water bath, opened, and the contents treated with 13 g of solid NaHCO3. The isobutylene was allowed to evaporate and the remaining mixture was concentrated in vacuo to dryness. The residue was suspended in 100 mL of water and extracted with EtOAc (4x40 mL). The combined EtOAc extracts were washed with 5% aqueous NaHCO3 (3x50 mL), dried over anhydrous MgSO4, and concentrated in vacuo to give an oil. This material was dissolved in 30 mL of ether and the solution was treated with 3.4 mL of 1M ethereal HCl. A white solid precipitated which was collected by filtration and dried in vacuo to afford 0.79 g (74%) of tert-butyl 3- methoxycarbonyl-L-phenylalaninate-HCl, m.p. 165°C (dec). Example 55 : /ert-Butyl N- benzyloxy ' .carbonyl ' . -5-O-tert-L-glutam- 1 -yl-3- methoxycarbonyl-L-phenylalaninate According to example 9, 0.79 g of /ert-butyl 3-methoxycarbonyl-L-phenylalaninate*HCl was coupled with 0.84 g of N-benzyloxycarbonyl-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.50 g of EDC (Aldrich) and 0.27 mL of N-methylmoφholine. The crude product was purified by flash chromatography on 85 g of silica gel (70:30 hexane:EtOAc) to afford 1.38 g (93%) of tert-butyl N-((benzyloxy)carbonyl)-5-0-tert-L- glutam-l-yl-3-methoxycarbonyl-L-phenylalaninate as a transparent glass. JH-ΝMR: (300 MHz, DMSO-dό) δ 8.26 (d, IH, J=7.5, ΝH), 7.80 (m, 2H, ΝH, aromatic CH), 7.43 (d, IH, J=7.7, aromatic CH), 7.44-7.23 (m, 7H, aromatic CH), 4.99 (m, 2H, PhCH 2 O), 4.38 (m, IH, methine), 4.01 (m, IH, methine), 3.83 (s, 3H, CO 2 CH3), 3.02 (m, 2H, ArCH 2 ), 2.19 (t, 2H, J=7.7, glu 4-CH 2 ), 1.90-1.61 (m, 2H, glu 3-CH 2 ), 1.38 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu). Example 56: tert-Butyl 5-O-tert-L-glutam- l-yl-3-methoxycarbonyl-L-phenylalaninate According to example 48, 1.38 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-L- glutam-l-yl-3-methoxycarbonyl-L-phenylalaninate was hydrogenolyzed using 0.23 g of 10% Pd(C) in 60 mL of MeOH. This afforded 1.0 g (94%) of tert-butyl 5-O-tert-L- glutam- l-yl-3-methoxycarbonyl-L-phenylalaninate as a thick, transparent oil. The product was carried on to the next step without purification. A sample of the corresponding HCl salt for ^H-NMR analysis was prepared by treating 7 mg of the product with excess 1M ethereal HCl and concentrating to dryness. JH-NMR: (300 MHz, DMSΟ-d6) δ 9.12 (d, IH, J=7.6, NH), 8.39 (br s, 3H, NH3 + ), 7.82 (m, 2H, aromatic CH), 7.60 (d, IH, J=7.5, aromatic CH), 7.46 (t, IH, J=6.9, aromatic CH), 4.40 (m, IH, methine), 3.85 (m, IH, methine), 3.83 (s, 3H, CΟ2CH3), 3.05 (d, 2H, J=8.4, ArCH 2 ), 2.33 (t, 2H, J=7.7, glu 4-CH 2 ), 1.97 (m, 2H, glu 3-CH 2 ), 1.37 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu). Example 57: tert-Butyl N-(4-fff2.4-diamino-6-pteridinyl')methvπmethylamino'.benzovπ- 5-O-tert-butyl-L-glutam- 1 -yl-3 -f methoxycarbonyl ' . -L-phenylalaninate According to example 49, 0.200 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.245 g of tert-butyl 5-O-tert-L-glutam- l-yl-3-methoxycarbonyl-L-phenylalaninate using 0.24 mL of DECP (Aldrich) and 0.26 mL of Et3Ν in 20 mL of DMF. The crude product was purified by flash chromatography on 130 g of silica gel (12:12:1 CH 2 Cl 2 :acetone:MeOH) to afford 0.297 g (73%) of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3- (methoxycarbonyl)-L-phenylalaninate as a yellow powder, m.p. 130°C (dec). Example 58 : N-f4-f f f 2.4-Diamino-6-pteridinyπmethvDmethylamino)benzoyiyL-glutam- 1 -yl-3-carboxy-L-phenylalanine According to example 50, 0.15 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3- (methoxycaιbonyl)-L-phenylal__ninate was treated with gaseous HCl in 20 mL of CH3NO2. After stirring the mixture at RT for lh, the mixture was concentrated in vacuo to dryness to give a yellow-orange solid. Analysis of this material by iH-NMR indicated complete loss of the tert-butyl groups of the starting material. HPLC (C18, 75:25:0.1 H 2 O:MeCN:TFA) indicated a single component at k'=3.50. The intermediate methyl ester was dissolved in 25 mL of 1:1 THF:H2θ, treated with 49 mg of LiOH H2O (Baker), and the solution was stirred at RT under N2 for 18h. Analysis by HPLC indicated no remaining k'=3.50 material and a single new component at k'=1.58. The solution was neutralized by addition of IN aqueous HCl. A yellow slurry resulted which was concentrated in vacuo to 2 mL and diluted with 20 mL of water. The precipitate was separated by centrifugation, washed with four cycles of aqueous suspension- centrifύgation-decantation, and lyophilized to afford 0.109 g (84%) of N-(4-(((2,4- diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3-carboxy-L- phenylalanine as a yellow-orange powder, m.p. 195°C (dec). HPLC: one peak on C18, k'=1.93, 78:22:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. JH-NMR: (300 MHz, DMSO-dό) δ 13.00-12.10 (br s, COOH), 8.58 (s, IH, pteridinyl 7-CH), 8.08 (d, IH, J=7.8, amide NH), 7.98 (d, IH, J=8.0, amide NH), 7.80 (s, IH, aromatic CH), 7.79-7.60 (m, 4H, NH2O), aromatic CH), 7.49 (br s, IH, NH2), 7.44 (d, IH, J=7.7, aromatic CH), 7.30 (t, IH, J=7.6, aromatic CH), 6.81 (d, 2H, J=9.0, aromatic CH), 6.63 (br s, 2H, NH2), 4.79 (s, 2H, pteridinyl-CH2), 4.41 (m, 2H, methines), 3.20 (s, 3H, N-CH3), 3.10 (m, IH, ArCHz), 2.97 (m, IH, A1-CH2), 2.23 (t, 2H, J=7.8, glu 4- CH 2 ), 2.00-1.73 (m, 2H, glu 3-CH2). Elemental Analysis: Calcd. for C30H31N9O8 I.5H2O (MW 672.65) C, 53.57; H, 5.09; N, 18.74. Found: C, 53.58; H, 5.11; N, 18.72. Mass Spectrum: (Ion Spray) 646 (M+H) + , 437, 340, 308, 175. UN Spectrum: (pH7 Buffer) λ max (ε) 258.7 (24300), 307.5 (25000), 372.7 (7880). λmin (ε) 244.0 (17900), 272.7 (16900), 346.2 (6440). Example 59: /ert-Butyl N- 4- f f 1 ,2-dihvdro-3-methyl- 1 -oxobenzoff.-quinazolin-9- yπmethyπaminoV2-fluorobenzoylV5-O-tert-butyl-L-glutam-l-yl-3-fmethoxycarbonylV L-phenylalaninate A 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.150 g of 4-(((l,2-dihydro-3-methyl-l-oxobenzo(f)quinazolin-9- yl)methyl)amino)-2-fluorobenzoic acid, 0.221 g of tert-butyl 5-O-tert-L-glutam- l-yl-3- methoxycarbonyl-L-phenylalaninate, 65 mg of 3-hydroxy-l,2,3-benzotriazin-4(3H)-one (Aldrich), and 24 mL of anhydrous DMF. The resulting light yellow solution was treated with 84 mg of EDC (Aldrich) and was stirred at RT under Ν2 for 48h. The DMF was removed in vacuo and the residue purified by flash chromatography on 150 g of silica gel (98:2->96:4 OfeCtø.MeOH) to afford 0.255 g (78%) of tert-butyl N-(4-(((l,2-dihydro- 3-methyl-l-oxobenzo(_^quinazolin-9-yl)methyl)aπ_ino)-2-fluorobenzoyl)-5-O-tert-butyl- L-glutam-l-yl-3-(methoxycarbonyl)-L-phenylalaninate as a white powder, m.p. 155- 157°C. Example 60: N- 4- ffl.2-Dihydro-3-methyl-l-oxobenzoff)quinazolin-9- yl)methyl aminoV2-fluorobenzoyπ-L-glutam-l-yl-3-carboxy-L-phenylalanine According to example 50, 0.219 g of tert-butyl N-(4-(((l,2-dihydro-3-methyl-l- oxobenzo(f)-quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-5-O-tert-butyl-L-glutam-l- yl-3-(methoxycarbonyl)-L-phenylalaninate was treated with gaseous HCl in 20 mL of CH3ΝΟ2. After removal of the CH3NO2 in vacuo, the residue was saponified with 66 mg of LiOH H2O (Baker) in 20 mL of 1 : 1 THF:H2θ for 18h. The solution was acidified to pH=2.5 by addition of IN HCl and the resulting precipitate was isolated and lyophilized in the usual manner to afford 0.162 g (84%) of N-(4-(((l,2-dihydro-3-methyl- 1 -oxobenzo(Qquinazo_-_n-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam- 1 -yl-3-carboxy- L-phenylalanine as a white powder, m.p. 187oC (dec). HPLC: one peak on C18, k"=4.29, 75:25:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. H-NMR: (300 MHz, DMSO-dό) δ 13.00-12.25 (br m, COOH, benzoquinazoline NH), 9.83 (s, IH, aromatic CH), 8.32 (d, IH, J=7.4, amide NH), 8.22 (d, IH, J=9.0, aromatic CH), 8.00 (d, IH, J=8.5, aromatic CH), 7.81 (s, IH, aromatic CH), 7.75 (d, IH, J=8.0, aromatic CH), 7.60 (m, 2H, aromatic CH), 7.49 (m, 3H, aromatic CH, 4-aminobenzoyl NH), 7.35 (m, 2H aromatic CH, amide NH), 6.52 (d, IH, J=8.8, aromatic CH), 6.39 (d, IH, J=15.2, aromatic CH), 4.57 (br s, 2H, benzoquinazoline 9-CH2), 4.45 (m, 2H, methines), 3.12 (m, IH, ArCH 2 ), 2.96 (m, IH, ArCH 2 ), 2.42 (s, 3H, CH3), 2.19 (m, 2H, glu 4-CH2), 2.01-1.70 (m, 2H, glu 3-CH2). Elemental Analysis: Calcd. for C36H32N5O9F I.7H2O (MW 728.30): C, 59.37; H, 4.90; N, 9.62. Found: C, 59.45; H, 4.93; N, 9.42. Mass Spectrum: (Ion Spray) 698 (M+H) + . UN Spectrum: (pH7 Buffer) λ m ax (ε) 265.3 (45500), 296.5 (26100), 348.0 (5530). λmin (ε) 244.4 (22600), 287.0 (25500), 340.1 (3540). Example 61: 2-Cyclopentylbenzyl alcohol A 300 mL Parr bomb was charged with 10.0 g of 2-iodobenzyl alcohol (Aldrich), 80 mL of toluene, 30 mL of cyclopentene (Aldrich), 2.60 g of tri-ortho-tolylphosphine (Aldrich), and 6.6 mL of Et3Ν. The mixture was deoxygenated by bubbling N2 through for 10 min and was then treated with 0.96 g of Pd(OAc)2 (Aldrich). The bomb was flushed with N2, sealed, and heated to 100°C for 18h. The vessel was cooled to RT, purged with N 2 and opened. The dark red reaction mixture was concentrated in vacuo to give a red-brown syrup. This material was dissolved in 200 mL of CH 2 C1 2 , washed with water (4x100 mL), dried over anhydrous MgSO4, and concentrated in vacuo to afford a brown oil. Analysis by tic (SiO 2 , 8:2 hexane:EtOAc) indicated two new major components at R =0.45 and 0.50, tri-o-tolylphosphine at Rf=0.90, and 4 minor components at Rf=0.60, 0.70, 0.85 and 0.0. The crude product was purified by flash chromatography (twice) on 200 g of silica gel (9:1 hexane:EtOAc) to afford 6.1 g (82%) of a mixture of the R =0.45 and 0.50 materials. This material was determined to be a mixture of double bond isomers by -1H-NMR. The product (6.0 g) was then hydrogenated at 40 psi in 40 mL of MeOH in the presence of 0.60 g of 5% Pt(C) (Aldrich) for 4h. The catalyst was removed by filtration through celite and the filtrate was concentrated in vacuo to afford 5.47 g (74% from 2-iodobenzyl alcohol) of 2-cyclopentylbenzyl alcohol as a light yellow-brown liquid. iH-NMR: (200 MHz, DMSO-d6) δ 7.40-7.07 (m, 4H, aromatic CH), 5.06 (t, IH, J=5.4, OH), 4.58 (d, 2H, J=5.4, ArCTfeO), 3.24 (m, IH, cyclopentyl methine), 1.97 (m, 2H, cyclopentyl CH2), 1.89-1.42 (m, 6H, cyclopentyl CH2). Example 62: 2-Cyclopentylbenzyl bromide According to example 17, 5.47 g of 2-cyclopentylbenzyl alcohol was treated with 1.03 mL of PB13 in 25 mL of anhydrous CH2CI2 to afford 6.9 g (93%) of 2-cyclopentylbenzyl bromide as a clear liquid. JH-NMR: (300 MHz, DMSO-dό) δ 7.40-7.24 (m, 3H, aromatic CH), 7.14 (m, IH, aromatic CH), 4.78 (s, 2H, ArCH2Br), 3.31 (m, IH, cyclopentyl methine), 2.05 (m, 2H, cyclopentyl CH2), 1.88-1.47 (m, 6H, cyclopentyl CH2). Example 63: f2R- 3S. 5S.-Benzyl 3-f3-cvclopentylbenzylV2-oxo-2.3-diphenyl-4- moφholinecarboxylate According to example 51, 1.47 g of benzyl (2R, 3S)-6-oxo-2,3-diphenyl-4- moφholinecarboxylate (Aldrich) was alkylated with 1.00 g of 2-cyclopentylbenzyl bromide using 3.99 mL of 1M sodium bis(trimethylsilyl)amide in THF (Aldrich). The crude product was purified by flash chromatography on 125 g of silica gel (8:2 hexane.EtOAc) to afford 1.07 g (52%) of (2R, 3S, 5S)-benzyl 3-(3-cycloρentylbenzyl)-2- oxo-2,3-diphenyl-4-moφholinecarboxylate as a white solid, m.p. 161-163°C. Example 64: 2-Cvclopentyl-L-phenylalanine According to example 53, 1.0 g of (2R, 3S, 5S)-benzyl 3-(3-cyclopentylbenzyl)-2-oxo- 5,6-diphenyl-4-moφholinecarbo>_ylate was hydrogenolyzed in 40 mL of 1 : 1 THF:MeOH using 0.23 g of PdC_2 to afford 0.373 g (76%) of 2-cyclopentyl-L-phenylalanine-HCl as an off-white powder, m.p. 175°C (dec). -1H-NMR: (200 MHz, DMSO-dό) δ 8.49 (br s, 3H, NH3+), 7.38-7.07 (m, 4H, aromatic CH), 3.92 (m, IH, methine), 3.18 (m, IH, cyclopentyl methine), 1.98 (m, 2H, cyclopentyl CH 2 ), 1.90-1.41 (m, 6H, cyclopentyl CH2). Example 65: tert-Butyl 2-cyclopentyl-L-phenylalaninate According to example 54, 0.23 g of 2-cyclopentyl-L-phenylalanineΗCl was treated with 25 mL of isobutylene in 20 mL of 1,4-dioxane in the presence of 0.13 mL of cone H2SO4. Work-up afforded a clear oil which was dissolved in 20 mL of ether. The solution was treated with 1 mL of 1 M ethereal HCl and concentrated in vacuo to dryness to afford 0.28 g (100%) of tert-butyl 2-cyclopentyl-L-phenylalaninateΗCl as a light yellow glass. 1H-NMR: (300 MHz, DMSO-d6) δ 8.59 (br s, 3H, NH3 + ), 7.28 (m, 2H, aromatic CH), 7.10 (m, 2H, aromatic CH), 3.91 (m, IH, methine), 3.30 (m, IH, ArCH2), 3.18 (m, IH, cyclopentyl methine), 2.99 (dd; IH; J=10.1, 12.4; ArCH2), l- 8 (m, 2H, cyclopentyl CH2), 1.89-1.46 (m, 6H, cyclopentyl CH 2 ), 1.19 (s, 9H, t-Bu). Example 66 : tert-Butyl N-f fbenzyloxy)cari?onvD-5-O-tert-butyl-L-glutam- 1 -yl-2- cvclopentyl-L-phenylalaninate According to example 9, 0.25 g of tert-butyl 2-cyclopentyl-L-phenylalaninateΗCl was coupled with 0.26 g of N-benzyloxycarbonyl-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.16 g of EDC (Aldrich) and 0.084 mL ofN-methylmoφholine. The crude product was purified by flash chromatography on 85 g of silica gel (8:2 hexane:EtOAc) to afford 0.355 g (76%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam- l-yl-2-cyclopentyl-L-phenylalaninate as a white solid, m.p. 95-96°C. Example 67: tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-2-cvclopentyl-L-phenylalaninate According to example 48, 0.355 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl- L-glutam-l-yl-2-cyclopentyl-L-phenylalaninate was hydrogenolyzed using 60 mg of 10% Pd(C) in 45 mL of MeOH. This afforded 0.236 g (85%) of tert-butyl 5-O-tert-butyl-L- glutam-l-yl-2-cyclopentyl-L-phenylalaninate as a thick, transparent oil. Ϊ H-ΝMR: (200 MHz, DMSΟ-d6) δ 8.23 (m, IH, NH), 7.34-7.00 (m, 4H, aromatic CH), 4.37 (m, IH, methine), 3.36-2.85 (m, 5H, ArCH2, cyclopentyl methine, NH2), 2.27-1.92 (m, 4H, glu 4-CH2, glu 3-CH2), 1.90-1.46 (m, 8H, cyclopentyl CH2), 1.39 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu). Example 68: tert-Butyl N*»*f4-fff2.4-diamino-6-pteridiny0methy0methylamino^benzoyl ' .- 5-O-tert-butyl-L-glutam- 1 -yl-2-cvclopentyl-L-phenylalaninate According to example 49, 0.189 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.236 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-2-cyclopentyl-L-phenylalaninate using 0.23 mL of DECP (Aldrich) and 0.24 mL of Et3Ν in 20 mL of DMF. The crude product was purified by flash chromatography on 85 g of silica gel (12:12:1 CH2Cl2:acetone:MeΟH) to afford 0.195 g (50%) of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-2-cyclopentyl-L- phenylalaninate as a yellow powder, m.p. 130-140°C. Example 69: N- 4-fff2.4-Diamino-6-pteridinvπmethyl)methylamino'.benzoylVL-glutam- 1 -yl-2-cyclopentyl-L-phenylalanine According to example 50, 0.167 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-2-cyclopentyl-L- phenylalaninate was treated with gaseous HCl in 15 mL of CH3ΝΟ2 for lh. The reaction mixture was concentrated in vacuo to dryness to give a yellow solid. This material was suspended in ether, collected by vacuum filtration, and dried in vacuo to afford 0.15 g (91%) ofN-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)- L-glutam-l-yl-2-cyclopentyl-L-phenylalanine as a light yellow powder, m.p. 155°C (dec). HPLC: one peak on C18, k'=3.25, 65:35:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. JH-NMR: (300 MHz, DMSO-d6) δ 13.25-12.95 (br s, COOH), 9.31 (s, IH, NH2), 9.09 (s, IH, NH2), 8.80-8.58 (br s, IN, NH2), 8.72 (s, IH, pteridinyl 7-CH), 8.24 (d, IH, J=8.0, amide NH), 8.01 (d, IH, J=8.0, amide NH), 7.74 (m, 3H, NH2(1), aromatic CH), 7.22 (d, IH, J=8.1, aromatic CH), 7.12 (m, 2H, aromatic CH), 6.93 (t, IH, J=7.4, aromatic CH), 6.82 (d, 2H, J=9.0, aromatic CH), 4.88 (s, 2H, pteridinyl-CH 2 ), 4.45 (m, IH, methine), 4.34 (m, IH, methine), 3.26-3.10 (m, 2H, ArCH2, cyclopentyl methine), 2.88 (m, IH, ArCH2), 2.23 (t, 2H, J=7.6, glu 4-CH 2 ), 2.06-1.40 (m, 10H, glu 3-CH2, cyclopentyl CH2). Elemental Analysis: Calcd. for C34H39N9O6- 1.7 HCl 2.2 H2O (MW 771.36): C, 52.94; H, 5.89; N, 16.34; CI, 7.81. Found: C, 52.57; H, 5.74; N, 16.13; CI, 7.52. Mass Spectrum: (Ion Spray) 670 (M+H) + , 613, 459, 391, 309, 275. UN Spectrum: (pH7 Buffer) λ m aχ (ε) 259,.5 (24000), 308.5 (24000), 373.0 (7450). λmin (ε) 241.0 (14400), 274.2 (16200), 346.5 (5840). Example 70: 3-Cyclopentylbenzyl alcohol According to example 61, 10.0 g of 3-iodobenzyl alcohol (Aldrich) was treated with 30 mL of cyclopentene using 0.96 g of Pd(OAc)2, 2.60 g of tri-ortho-tolylphosphine, and 6.6 mL of Et3Ν. The resulting olefin mixture was hydrogenated for 2.5h using 0.62 g of 5% Pt(C) in MeOH to afford 5.77 g (76%) of 3-cyclopentylbenzyl alcohol as a light yellow liquid. iH-NMR: (200 MHz, DMSO-dό) δ 7.32-7.17 (m, 2H, aromatic CH), 7.15 (m, 2H, aromatic CH0, 5.14 (t, IH, J=5.5, OH), 4.48 (d, 2H, J=5.6, ArQfeO), 2.96 (m, IH, cyclopentyl methine), 2.01 (m, 2H, cyclopentyl CH2), 1.88-1.42 (m, 6H, cyclopentyl CH 2 ). Example 71: 3-Cyclopentylbenzyl bromide According to example 17, 5.77 g of 3-cyclopentylbenzyl alcohol was treated with 1.09 mL of PB13 in 25 mL of anhydrous CH2CI2 to afford 7.85 g (100%) of 3- cyclopentylbenzyl bromide as a light yellow liquid. H-NMR: (300 MHz, DMSO-dό) δ 7.37-7.14 (m, 4H, aromatic CH), 4.68 (s, 2H, ArCH2Br), 2.96 (m, IH, cyclopentyl methine), 2.00 (m, 2H, cyclopentyl CH2), 1.84- 1.42 (m, 6H, cyclopentyl CH2). Example 72: (2R. 3S. 5S.-Benzyl 3-f3-cvclopentylbenzylV2-oxo-2.3-diphenyl-4- moφholinecarboxylate A dry 500 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 1.47 g of benzyl (2R, 3_S)-6-oxo-2,3-diphenyl-4-moφholinecarboxylate (Aldrich), 1.00 g of 3-cyclopentylbenzyl bromide , and 80 mL of anhydrous THF. The mixture was gently heated with stirring to dissolve the solid starting material and the resulting solution was cooled to -78°C. The solution was treated with 4.0 mL of 1M sodium bis(trimethylsilyl)amide in THF (Aldrich) by slow addition via syringe through a rubber septum. After stirring at -78°C for 4h, the solution was mixed with 80 mL of water and worked-up according to example 51. The crude product was purified by flash chromatography on 85 g of silica gel (85:15 hexane.EtOAc) to afford 1.44 g (70%) of (2R, 3S, 5S)-Benzyl 3-(3-cyclopentylbenzyl)-2-oxo-2,3-diphenyl-4- moφholinecaiboxylate as a white powder, m.p. 82-84 °C. Example 73: 3-Cyclopentyl-L-phenylalanine According to example 53, 1.37 g of (2R, 3S, 5S)-benzyl 3-(3-cyclopentylbenzyl)-2-oxo- 5,6-diphenyl-4-moφholinecarboxylate was hydrogenolyzed in 80 mL of 1:1 THF:MeOH using 0.31 g of PdCl2 to afford 0.63 g (93%) of 3-cyclopentyl-L-phenylalanine HCl as a white powder, m.p. >200°C. JH-NMR: (200 MHz, DMSO-dό) δ 8.39 (br s, 3H, NH3 -), 7.31-7.12 (m, 3H, aromatic CH), 7.08 (d, IH, J=7.3, aromatic CH), 4.17 (m, IH, methine), 3.11 (d, 2H, J=6.1, ArCH2), 2.96 (m, IH, cyclopentyl methine), 1.99 (m, 2H, cyclopentyl CH2), 1.88-1.45 (m, 6H, cyclopentyl CH2). Example 74: tert-Butyl 3-cyclopentyl-L-phenylalaninate According to example 54, 0.50 g of 3-cyclopentyl-L-phenylalanine*HCl was treated with 25 mL of isobutylene in 20 mL of 1,4-dioxane in the presence of 0.3 mL of cone H2SO4. Work-up followed by acidification of the free amine with ethereal HCl afforded 0.49 g (82%) of tert-butyl 3-cyclopentyl-L-phenylalaninateΗCl as a white powder, m.p. 154-156°C. Example 75: tert-Butyl N-ffbe_-zyloxy rt>onyiy5-O-tert-butyl-L-glutam-l-yl-3- cvclopentyl-L-phenylalaninate According to example 9, 0.49 g of tert-butyl 3-cyclopentyl-L-phenylalaninateΗCl was coupled with 0.51 g ofN-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.30 g of EDC (Aldrich) and 0.16 mL of N-methylmoφholine. The crude product was purified by flash chromatography on 80 g of silica gel (8:2 hexane:EtOAc) to afford 0.75 g (82%) of /ert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam- 1 -yl-3-cyclopentyl-L- phenylalaninate as a thick transparent oil. *H-ΝMR: (300 MHz, DMSO-dό) δ 8.22 (d, IH, J=7.3, NH), 7.33 (m, 6H, aromatic CH), 7.20-6.97 (m, 3H, aromatic CH, NH), 5.01 (m, 2H, PhCH2θ), 4.33 (m, IH, methine), 4.05 (m, IH, methine), 2.91 (m, 3H, ArCH2, cyclopentyl methine), 2.20 (t, 2H, J=7.7, glu 4-CH2), 1.97 (m, 2H, glu 3-CH2), 1.88-1.43 (m, 8H, cyclopentyl CH2), 1.38 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu). Example 76: tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-3-cvclopentyl-L-phenylalaninate According to example 48, 0.75 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L- glutam-l-yl-3-cyclopentyl-L-phenylalaninate was hydrogenolyzed using 0.12 g of 10% Pd(C) in 60 mL of MeOH. This afforded 0.58 g (99%) of tert-butyl 5-O-tert-butyl-L- glutam-l-yl-3-cyclopentyl-L-phenylalaninate as a transparent oil. A sample of the corresponding HCl salt for ^H-ΝMR analysis was prepared by treating 10 mg of the product with excess IN ethereal HCl and concentrating to dryness. H-ΝMR: (300 MHz, DMSO-dό) δ 8.99 (d, IH, J=7.0, ΝH). 8.32 (br s, 3H, ΝH3+), 7.24-7.01 (m, 4H, aromatic CH), 4.38 (m, IH, methine), 3.85 (m, IH, methine), 2.93 (m, 3H, ArCH2, cyclopentyl methine), 2.34 (t, 2H, J=7.7, glu 4-CH2), 1.96 (m, 4H, glu 3- CH 2) cyclopentyl CH 2 ), 1.82-1.44 (m, 6H, cyclopentyl CH 2 ), 1.40 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu). Example 77: tert-Butyl 5-O-tert-butyl-N-(4-fff2.4-diamino-6- pteridinvπmethvπmethylamino ' .benzoylVL-glutam-l-yl-3-cvclopentyl-L-phenylalaninate According to example 49, 0.200 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.25 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate using 0.24 mL of DECP (Aldrich) and 0.26 mL of Et3Ν in 20 mL of DMF. The crude product was purified by flash chromatography on 150 g of silica gel (7:7:1 CH 2 Cl 2 :acetone:MeΟH) to afford 0.195 g (47%) of tert-butyl 5-O-tert-butyl-N-(4- (((2,4-di__mino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3 -cyclopentyl- L-phenylalaninate as a yellow powder, m.p. 115-120°C. Example 78 : N- 4-f f 2.4-Di-ffliino-6-pteridinvDmethvDmethylamino)benzoyD-L-glutam- 1 -yl-3-cyclopentyl-L-phenylalanine According to example 50, 0.18 g of tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate was treated with gaseous HCl in 20 mL of CH3ΝΟ2 for 1.25h. Work-up and isolation in the usual manner afforded 0.135 g (84%) of N-(4-(((2,4-di__mino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3-cyclopentyl-L-phenylalanine as a yellow-orange powder, m.p. 170°C (dec). HPLC: one peak on C18, k'-=2.86, 65:35:0.1 H 2 O:MeCΝ:TFA, flow rate=l mL/min. *H-NMR: (300 MHz, DMSO-dό) δ 12.90-11.85 (br m, COOH), 8.58 (s, IH, pteridinyl 7-CH), 8.04 (d, IH, J=7.7, amide NH), 8.00 (d, IH, J=8.1, amide NH), 7.72 (br s, IH, NH 2 ), 7.71 (d, 2H, J=8.8, aromatic CH), 7.53 (br s, IH, NH 2 ), 7.08-6.93 (m, 4H, aromatic CH), 6.81 (d, 2H, J=8.9, aromatic CH), 6.69 (br s, 2N, NH ), 4.79 (s, 2H, pteridinyl-CH 2 ), 4.40 (m, 2H, methines), 3.22 (s, 3H, N-CH3), 3.01 (m, IH, ArCH 2 ), 2.94-2,.75 (m, 2H, ArCH 2 , cyclopentyl methine), 2.24 (t, 2H, J=7.7, glu 4-CH 2 ), 2.03- 1.76 (m, 4H, glu 3-CH 2 , cyclopentyl CH 2 ), 1.73-1.35 (m, 6H, cyclopentyl CH 2 ). Elemental Analysis: Calcd. for C34H39N9O6 I 6 H 2 O (MW 698.56): C, 58.46; H, 6.09; N, 18.05. Found: C, 58.50; H, 6.08; N, 18.10. Mass Spectrum: (FAB) 670 (M+H) + , 613, 437, 309, 275. UN Spectrum: (pH7 Buffer) λ max (ε) 259.7 (23300), 308.3 (23500), 371.7 (7030). λmin (ε) 241.0 (13500), 274.8 (15300), 346.1 (5260). Example 79: 2-Cyclohexylbenzyl alcohol According to example 61, 10.0 g of 2-iodobenzyl alcohol was treated with 25 mL of cyclohexene (Aldrich) using 0.96 g of Pd(OAc)2, 2.60 g of tri-ortho-tolylphosphine, and 6.6 mL of Et3Ν at 110°C for 48h. The resulting olefin mixture was hydrogenated for 6.5h using 0.36 g of 5% Pt(C) in MeOH to afford 3.37 g (42%) of 2-cyclohexyl benzyl alcohol as a light yellow liquid. 1 H = NMR: (300 MHz, DMSO-dό) δ 7.33 (d, IH, J=7.4, aromatic CH), 7.27-7.08 (m, 3H, aromatic CH), 5.03 (t, IH, J=5.4, OH), 4.34 (d, 2H, J=5.4, ArCH O), 2.76 (m, IH, cyclohexyl methine), 1.85-1.61 (m, 5H, cyclohexyl CH2), 1.48-1.22 (m, 5H, cyclohexyl CH 2 ). Example 80: 2-Cyclohexylbenzyl bromide According to example 17, 3.36 g of 2-cyclohexylbenzyl alcohol was treated with 0.59 mL of PB13 in 25 mL of anhydrous CH 2 C1 2 to afford 3.90 g of crude product (90% pure by ^H-NMR). This material was vacuum distilled via short path (1.0 mmHg, 117-120°C) to afford 3.54 g (79%) of 2-cyclohexylbenzyl bromide as a clear liquid. iH-NMR: (300 MHz, DMSO-dό) δ 7.39 (d, IH, J=5.7, aromatic CH), 7.29 (m, 2H, aromatic CH), 7.13 (m, IH, aromatic CH), 4.76 (s, 2H, ArCH2Br), 2.86 (m, IH, cyclohexyl methine), 1.83-1.63 (m, 5H, cyclohexyl CH 2 ), 1.53-1.20 (m, 5H, cyclohexyl CH2). Example 81: (2R. 3S. 5SVtert-Butyl 3-f2-cvclohexylbenzylV2-oxo-2.3-diρhenyl-4- moφholinecarboxylate To a dry 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was added a solution of 1.47 g of tert-butyl (2R, 3S)-(-)-6-oxo-2,3-diphenyl-4- moφholinecarboxylate (Aldrich) in 30 mL of THF and a solution of 1.00 g of 2- cyclohexylbenzyl bromide in 8 mL of THF. Both solutions had been dried over 3 A molecular sieves for 24h. Anhydrous THF was used for rinse (30 mL) to give a total solution volume of 68 mL. The resulting solution was cooled to -78°C and was treated with 4.15 mL of IM sodium bis-(trimethylsilyl) amide in THF (Aldrich) by slow addition via syringe through a rubber septum. After stirring at -78°C for 3.5h, the reaction mixture was mixed with 80 mL of water and subjected to rotary evaporation to remove THF. The resulting mixture was extracted with EtOAc (4x50 mL). The combined EtOAc extracts were washed with saturated aqueous NaCl (2x70 mL), dried over anhydrous MgSO4 and concentrated in vacuo to afford a white solid. Purification of this material by flash chromatography on 200 g of silica gel (8:2 hexane:EtOAc) afforded 1.2 g (58%) of (2R, 3S, 5S)-tert-butyl 3-(2-cyclohexylbenzyl)-2-oxo-2,3-diphenyl-4- moφholinecarboxylate as a white, crystalline solid, m.p. 195-198°C. Example 82: f2R. 3S. 5_Sy6-Oxo-2.3-diphenyl-5-f2-cvclohexylbenzvDmoφholine A dry 50 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 2.10 g of (2R, 3S, 5S)-tert-butyl 3-(2-cyclohexylbenzyl)-2-oxo-5,6- diphenyl-4-moφholinecarboxylate and 33 mL of anhydrous CH C1 2 . The solution was stirred at RT and treated with 3.3 mL of TFA. After 3.5h, tic (SiO , 85:15 hexane:EtOAc) indicated no remaining starting material, a major new component at R =0.45, and two minor components at Rf=0.68 and 0.70. The solution was neutralized by addition of 9 mL of Et3N and was concentrated in vacuo to give a thick oil. This material was dissolved in 80 mL of CH 2 C1 2 , washed with water (3x60 mL) followed by 5% aqueous NaHCO3 (3x60 mL) and dried over anhydrous MgSO4. Drying agent was removed by filtration and the filtrate concentrated in vacuo to afford a white solid. Purification of the crude product by flash chromatography on 150 g of silica gel (8:2 hexane:EtOAc) followed by recrystallization from EtOH-H 2 O afforded 1.27 g (75%) of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(2-cyclohexylbenzyl)moφholine as a white crystalline solid, m.p. 159-16Q C. Example 83: 2-Cyclohexyl-L-phenylalanine According to example 53, 1.17 g of (2R, 35, 5SV6-oxo-2,3-diphenyl-5-(2- cyclohexylbenzyl)moφholine was hydrogenolyzed in 60 mL of 1 : 1 THF:EtOH using 0.34 g of PdCl2 to afford 0.83 g of 2-cyclohexyl-L-phenylalanine*HCl as a fluffy white solid, m.p. 140 C (dec). Example 84: tert-Butyl 2-cvclohexyl-L-phenylalaninate According to example 54, 0.73 g of 2-cyclohexyl-L-phenylalanine HCl was treated with 25 mL of isobutylene in 25 mL of 1,4-dioxane in the presence of 0.4 mL of cone H 2 SO4. Work-up followed by acidification of the free amine with ethereal HCl afforded 0.70 g (80%) of tert-butyl 2-cyclohexyl-L-phenylalaninate*HCl as a yellow foam. JH-NMR: (300 MHz, DMSO-dό) δ 8.53 (br s, 3H, NH3 + ), 7.27 (m, 2H, aromatic CH), 7.10 (m, 2H, aromatic CH), 3.86 (m, IH, methine), 3.24 (m, IH, ArCH 2 ), 2.97 (m, IH, ArCH2), 2.72 (m, IH, cyclohexyl methineJ), 1.86-1.60 (m, 5H, cyclohexyl CH2), 1.56- 1.30 (m, 5H, cyclohexyl CH 2 ), 1.19 (s, 9H, t-Bu). Example 85: tert-Butyl N-ffbenzyloxy.carbonylV5-O-tert-butyl-L-glutam-l-yl-2- cyclohexyl-L-phenyalaninate According to example 9, 0.70 g of tert-butyl 2-cyclohexyl-L-phenylalaninateΗCl was coupled with 0.70 g of N-Cbz-L-glutamic acid-γ-tert-butyl ester (Sigma) using 0.42 g of EDC (Aldrich) and 0.23 mL of N-methylmoφhnoline. The crude product was purified by flash chromatography on 150 g of silica gel (8:2 hexane.EtOAc) to afford 0.97 g (76%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam- 1 -yl-2-cyclohexyl- L-phenyalaninate as a white foam. iH-ΝMR: (300 MHz, DMSO-dό) δ 8.32 (d, IH, J=7.6, ΝH), 7.40-6.97 (m, 10H, aromatic CH, ΝH), 5.00 (m, 2H, PI1CH2O), 4.26 (m, IH, methine), 4.04 (m, IH, methine), 3.07 (m, IH, ArCH 2 ), 2.93-2.70 (m, 2H, ArCH2, cyclohexyl methine), 2.19 (t, 2H, J=7.7, glu 4-CH2), 1.90-1.62 (m, 7H, glu 3-CH2, cyclohexyl CH2), 1.56-1.33 (m, 5H, cyclohexyl CH2), 1 8 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu). Mass Spectrum: (CI, CH4) 623 (M+H) + , 567, 539, 511, 489, 433, 193. Example 86: tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-2-cyclohexyl-L-phenyalaninate According to example 48, 0.97 g of tert-butyl N-((benzyloxy)carbonyl)-5-Ο-tert-butyl-L- glutam-l-yl-2-cyclohexyl-L-phenyalaninate was hydrogenolyzed using 0.16 g of 10% Pd(C) in 50 mL of MeOH. This afforded 0.76 g (100%) of tert-butyl 5-O-tert-butyl-L- glutam-l-yl-2-cyclohexyl-L-phenyalaninate as a thick transparent oil. A sample of the corresponding HCl salt for -1H-ΝMR analysis was prepared by treating 10 mg of the product with excess IM ethereal HCl and concentrating to dryness. JH-NMR: (300 MHz, DMSO-dό) δ 9.05 (d, IH, J=7.5, NH), 8.24 (br s, 3H, NH3+), 7.20 (m, 3H, aromatic CH), 7.07 (t, IH, J=7.1, aromatic CH), 4.28 (m, IH, methine), 3.83 (m, IH, methine), 3.08 (m, IH, ArCH2), 2.91 (m, IH, ArCH2), 1 78 (m, IH, cyclohexyl methine), 2.34 (m, 2H, glu 4-CH2), 2.00 (m, 2H, glu 3-CH2), 1.89-1.60 (m, 5H, cyclohexyl CH2), 1.57-1.20 (m, 5H, cyclohexyl CH2), 1.40 (s, 9H, t-Bu), 1.33 (s, 9H, t-Bu). Example 87: tert-Butyl N-f4-fff2.4-diamino-6-pteridinvπmethvπmethylamino)benzovπ- 5-O-tert-butyl-L-glutam- 1 -yl-2-cvclohexyl-L-phenylalaninate According to example 49, 0.247 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.35 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-2-cyclohexyl-L-phenyalaninate using 0.30 mL of DECP (Aldrich) and 0.32 mL of Et3Ν in 25 mL of DMF. The crude product was subjected to flash chromatography twice (150 g Siθ2, 7:7:1 CH2Cl2:acetone:MeOH; 150 g Siθ2, 95:5 OftCfc.MeOH) to afford 0.297 g (57%) of tert-butyl N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl- L-glutam-l-yl-2-cyclohexyl-L-phenylalaninate as a yellow powder, m.p. 135-140°C. Example 88 : N-f4- 2.4-Diamino-6-pteridinyπmethyl ' .methylamino'.benzovπ-L-glutam- 1 -yl-2-cyclohexyl-L-phenylalanine According to example 50, 0.271 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-Ο-tert-butyl-L-glutam-l-yl-2-cyclohexyl-L- phenylalaninate was treated with gaseous HCl in 20 mL of CH3ΝΟ2 for 1.5 h. Work-up and isolation in the usual manner afforded 0.191 g (79%) of N-(4-(((2,4-diaπ_ino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glut am- 1 -yl-2-cyclohexyl-L-phenylalanine as a yellow-orange powder, m.p. 175°C (dec). HPLC: one peak on C18, k'=4.05, 65:35:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. iH-NMR: (300 MHz, DMSO-dό) δ 12.75-11.90 (br s, COOH), 8.58 (s, IH pteridinyl 7-CH), 8.20 (d, IH, J=7.9, amide NH), 7.98 (d, IH, J=8.1, amide NH), 7.79 (br s, IH, NH2), 7.72 (d, 2H, J=8.9, aromatic CH), 7.59 (br s, IH, NH2), 7.19 (m, IH, aromatic CH), 7.08 (m, 2H, aromatic CH), 6.92 (m, IH, aromatic CH), 6.83 (d, 2H, J=8.9, aromatic CH), 6.75 (br s, 2H, NH2), 4.80 (s, 2H, pteridinyl-CH2), 4.43 (m, IH, methine), 4.31 (m, IH, methine), 3.21 (s, 3H, N-CH3), 3.12 (m, IH, ArCH2), 3.,92-3.70 (m, 2H, A1-CH2, cyclohexyl methine), 2.23 (t, 2H, J=7.7, glu 4-CH2), 2.05-1.60 (m, 7H, glu 3-CH2, cyclohexyl CH2), 1.50-1.16 (m, 5H, cyclohexyl CH2). Elemental Analysis: Calcd. for C35H4lN9θό- 1.5 H2O (MW 710.79): C, 59.14; H, 6.24; N, 17.74. Found: C, 59.11; H, 6.20; N, 17.73. Mass Spectrum: (FAB) 684 (M+H) + , 613, 510, 385, 309. UN Spectrum: (pH7 Buffer) λmax (ε) 259.7 (22300), 308.8 (22700), 374.0 (6850). λmin (ε) 241.7 (12800), 275.2 (14700), 346.3 (5170). Example 89: 3-tert-Butylbenzyl bromide A dry 100 mL 3-necked flask equipped with a nitrogen inlet, a condenser, a thermometer, and a magnetic stirrer was charged with 2.0 g of 3-tert-butyltoluene (Wiley Organics, Coshocton, OH 43812), 2.40 g of N-bromosuccinimide (Aldrich), and 30 mL of CCI4. The mixture was treated with 0.16 g of benzoyl peroxide (Aldrich) and heated at 60°C under Ν2. Careful temperature control is important to minimize by-product formation. After lh at 60°C the reaction mixture was cooled to 0°C, filtered to remove succinimide, and the filtrate was concentrated in vacuo to afford a light yellow liquid. Analysis of this material by tic (Siθ2, hexane) indicated a major new component at R =0.50, a minor component at R =0.55, and a trace of 3-tert-butyltoluene at Rf=0.75. The crude product was purified by flash chromatography on 100 g of silica gel (hexane) to afford 1.06 g (35%) of 3-tert-butylbenzyl bromide as a clear liquid. iH-NMR: (300 MHz, DMSO-dό) δ 7.47 (s, IH, aromatic CH), 7.39-7.21 (m, 3H, aromatic CH), 4.69 (s, 2H, ArCH2Br), 1.27 (s, 9H, t-Bu). Example 90: f2R. 3S. 5S.-tert-Butyl 3-f3-tert-butylbenzylV2-oxo-2.3-diρhenyl-4- moφholinecarboxylate According to example 81, 1.63 g of tert-butyl (2R, 3S)-(-)-6-oxo-2,3-diphenyl-4- moφholinecarboxylate (Aldrich) was alkylated with 1.0 g of 3-tert-butylbenzyl bromide in 70 mL of anhydrous THF using 4.62 mL of IM sodium bis(trimethylsilyl)amide in THF (Aldrich). The crude product was purified by flash chromatography on 150 g of silica gel (9:1 hexane: EtOAc) to afford 1.84 g (80%) of (2R, 3S, 5S)-tert-butyl 3-(3- tert-butylbenzyl)-2-oxo-2,3-diphenyl-4-moφholinecarboxylate as a white foam, m.p 65- 90°C. Example 91: (2R. 3S. 5S -6-Oxo-2.3-diphenyl-5-f3-tert-butylbenzvnmoφholine According to example 82, 1.79 g of (2R, 3S, 5S)-tert-butyl 3-(3-tert-butylbenzyl)-2-oxo- 5,6-diphenyl-4-moφholinecarboxylate was treated with 3 mL of TFA in 30 mL of CH 2 C_2 for 3.4h. Neutralization with 7.7 mL of Et3N and work-up in the usual manner gave a thick oil. Purification of the crude material by flash chromatography on 125 g of silica gel (75:25 hexane.EtOAc) afforded 0.90 g (63%) of (2R, 3S, 5S)-6-oxo-2,3- diphenyl-5-(3-tert-butylbenzyl)moφholine as a white crystalline solid, m.p. 136-137°C. Example 92: 3-tert-Butyl-L-phenylalanine According to example 53, 0.89 g of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert- butylbenzyl)moφholine was hydrogenolyzed in 40 mL of 1 : 1 THF:EtOH using 0.28 g of PdCl2 to afford 0.57 g (99%) of 3-tert-butyl-L-phenylalanine-HCl as a white powder, m.p. >250°C. ^-NMR: (300 MHz, DMSO-dό) 8.42 (br s, 3H, NH3 + ), 7.26 (m, 3H, aromatic CH), 7.08 (dd; IH; J=7.0, 1.3; aromatic CH), 4.12 (m, IH, methine), 3.12 (d, 2H, J=6.1, ArCH2), 1.27 (s, 9H, t-Bu). Example 93: tert-Butyl 3-tert-butyl-L-phenylalaninate According to example 54, 0.50 g of 3-tert-butyl-L-phenylalanineΗCl was treated with 30 mL of isobutylene in 25 mL of 1,4-dioxane in the presence of 0.4 mL of cone H2SO4. Work-up in the usual manner followed by acidification of the free amine with ethereal HCl afforded 0.53 g (87%) of tert-butyl 3-tert-butyl-L-phenylalaninate-HCl as a light yellow foam. *H-NMR: (300 MHz, DMSO-d6) δ 8.48 (br s, 3H, NH3 " ), 7.36-7.20 (m, 3H, aromatic CH), 7.08 (d, IH, J=7.1, aromatic CH), 4.16 (m, IH, methine), 3.19 (dd; IH; J=13.9, 5.7; ArCH2), 2.95 (dd; IH; J=13.9, 8.6; ArCH2), 1.28 (s, 9H, t-Bu), 1.27 (s, 9H, t-Bu). Example 94: tert-Butyl N-ffbenzyloxy ' )carbonyl>-5-O-tert-butyl-L-glutam-l-yl-3-tert- butyl-L-phenylalaninate According to example 9, 0.52 g of tert-butyl 3-tert-butyl-L-phenylalaninate-HCl was coupled with 0.56 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.33 g of EDC (Aldrich) and 0.18 mL of Ν-methylmoφholine. The crude product was purified by flash chromatography on 110 g of silica gel (75:25 hexane:EtOAc) to afford 0.77 g (78%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam- 1 -yl-3-tert-butyl- L-phenylalaninate as a thick transparent oil. JH-ΝMR: (200 MHz, DMSO-dό) δ 8.28 (d, IH, J=7.3, NH), 7.42-7.13 (m, 9H, aromatic CH, NH), 7.04 (d, IH, J=6.6, aromatic CH), 5.02 (s, 2H, PhCH2θ), 4.37 (m, IH, methine), 4.07 (m, IH, methine), 2.96 (d, 2H, J=7.6, ArCH2), 2.21 (t, 2N, J=7.7. glu 4-CH2) . 1.97-1.60 (m, 2H, glu 3-CH2), 1.39 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu), 1.27 (s, 9H, t-Bu). Example 95: tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-3-tert-butyl-L-phenylalaninate According to example 48, 0.76 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L- glutam-l-yl-3-tert-butyl-L-phenylalaninate was hydrogenolyzed using 0.13 g of 10% Pd(C) in 60 mL of MeOH. This afforded 0.58 g (99%) of tert-butyl 5-O-tert-butyl-L- glutam-l-yl-3-tert-butyl-L-phenylalaninate as a thick transparent oil. A sample of the corresponding HCl salt for -1H-ΝMR analysis was prepared by treating 10 mg of the product with excess IN ethereal HCl and concentrating to dryness. *H-NMR: (300 MHz, DMSO-dό) δ 8.97 (d, IH, J=7.0, NH), 8.27 (br s, 3H, NH3 + ), 7.31-7.15 (m, 3H, aromatic CH), 7.06 (d, IH, J=6.8, aromatic CH), 4.38 (m, IH, methine), 3.82 (m, IH, methine), 2.96 (d, 2H, J=7.2, ArCH2), 2.33 (m, 2H, glu 4-CH2), 1.98 (m, 2H, glu 3-CH2), 1.38 (s, 9H, t-Bu), 1.29 (s, 9H, t-Bu), 1.26 (s, 9H, t-Bu). Example 96: tert-Butyl N-(4-fff2.4-diamino-6-pteridinyl)methyπmethylamino.benzoyl ' )- 5-O-tert-butyl-L-glutam- 1 -yl-3 -tert-butyl-L-phenylalaninate According to example 49, 0.223 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.30 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3-tert-butyl-L-phenylalaninate using 0.27 mL of DECP (Aldrich) and 0.29 mL of Et3Ν in 25 mL of DMF. The crude product was subjected to flash chromatography twice (180 g Siθ2, 7:7:1 CH2Cl2:acetone:MeΟH; 185 g Siθ2, 95:5 CH2C_2:MeΟH) to afford 0.323 g (71%) of tert-butyl N-^4-(((2,4-dian_ino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl- L-glutam-l-yl-3-tert-butyl-L-phenylalaninate as a yellow powder, m.p. 128-135°C. Example 97 : N-f4- f f 2.4-Diamino-6-pteridinv0methy0methylamino ' .benzoyl L-glutam- l-yl-3-tert-butyl-L-phenylalanine fProcedure A) For Procedure B to prepare this compound, see Example 130. According to example 50, 0.303 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam- 1 -yl-3 -tert-butyl-L- phenylalaninate was treated with gaseous HCl in 40 mL of CH3ΝO2 for 1.5 h. Work-up and isolation in the usual manner afforded 0.215 g (79%) of N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3-tert-butyl-L-phenylalanine as a yellow powder, m.p. 183°C. HPLC: one peak on C18, k'=2.54, 70:30:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. 1H-NMR: (300 MHz, DMSO-dό) δ 12.90-11.80 (br s, COOH), 8.59 (s, IH, pteridinyl 7-CH), 8.08 (d, IH, J=7.6, amide NH), 7.98 (d, IH, J=8.0, amide NH), 7.89 (br s, IH, NH 2 ), 7.70 (d, 2H, J=8.9, aromatic CH), 7.69 (br s, IH, NH2), 7.21 (s, IH, aromatic CH), 7.16 (d, IH, J=7.7, aromatic CH), 7.08 (t, IH, J=7.6, aromatic CH), 6.99 (d, IH, J=7.4, aromatic CH), 6.81 (m, 4H, aromatic CH, NH2), 4.79 (s, 2H, pteridinyl CH2), 4.43 (m, 2H, methines), 3.21 (s, 3H, N-CH3), 3.03 (m, IH, ArCH2), 2.91 (m, IH, ArCH2), 2.24 (t, 2H, J=7.8. glu 4-CH2), 2.04-1.74 (m, 2H, glu 3-CH2), 1.20 (s, 9H, t- Bu). Elemental Analysis: Calcd. for C33H39N9O6 I.8 H2O (MW 690.16): C, 57.43; H, 6.22; N, 18.27. Found: C, 57.34; H, 6.07; N, 18.26. Mass Spectrum: (FAB) 658 (M+H) + , 459, 307. UN Spectrum: (pH7 Buffer) λ ma χ (ε) 259.4 (22800), 308.1 (23000), 372.5 (7040). λmin (ε) 241.0 (13400), 274.6 (15200), 345.2 (5380). Example 98 : N-f tert-Butoxy ' ) carbonyπ-4-fbisf 2-chloroethyl ' . amino L-phenylalanine A dry 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.10 g of 4'-(bis(2-chloroethyl)amino)-L-phenylalanineΗ2θ (Sigma), 14 mL of 1 : 1 DMF:CH3CΝ, and 0.19 mL of Et3N. The mixture was treated with 91 mg of 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-ON, Fluka) and stirred at RT under N2- After 18h, the solid starting material had dissolved giving a light yellow solution which was concentrated in vacuo to dryness. Analysis of the residue by tic (Siθ2, 9:1 CHCl3:MeOH) indicated a major new component at Rf=0.65, BOC-ON derived components at Rf=1.0 and 0.71 and two trace components at Rf=0.60 and 0.35. The crude product was purified by flash chromatography on 60 g of silica gel (9: 1 CHCl3:MeOH) to afford 0.10 g (80%) ofN-((tert-butoxy)carbonyl)-4-(bis(2- chloroethyl)amino)-L-phenylalanine as light yellow glass. JH-NMR: (300 MHz, DMSO-dό) δ 7.04 (d, 2H, J=8.6, aromatic CH), 6.62 (d, 2H, J=8.5, aromatic CH), 6.46 (br s, IH, NH), 3.87 (m, IH, methine), 3.67 (s, 8H, N (CH2CH2C 2), 2.90 (m, IH, ArCH2), 2.73 (m, IH, ArCH2), 1.33 (s, 9H, t-Bu). * Example 99: tert-Butyl N ftert-butoxy arbonyπ-4-fbisf2-chloroethvπaminoVL- phenylalanyl-f3-cvclopentylVL-phenylalaninate A 50 mL round bottomed flask equipped with a magnetic stirrer was charged with 96 mg of N*^(tert-butoxy)c___rbonyl)*4-(bis(2-cWoroethyl)__mino)-L-phenylalanine 77 mg of tert- butyl 3-cyclopentyl-L-phenylalaninateΗCl, and 10 mL of CH 2 C1 . The solution was cooled to 0°C and treated with 48 mg of EDC (Aldrich) followed by 0.026 mL of N- methylmoφholine. The reaction mixture was stirred at 0°C for 20 min, allowed to warm to RT, and stirred for an additional 2h. Analysis of the solution by tic (Siθ2, 7:3 hexane:EtOAc) indicated a major new component at Rf=0.54 and four minor components at Rf=0.4, 0.37, 0.16 and 0.0. The solution was concentrated in vacuo to dryness and the residue was subjected to flash chromatography on 50 g of silica gel to afford 73 mg (46%) of tert-butyl N-((tert-butoxy)carbonyl)-4-(bis(2-chloroethyl)amino)- L-phenylalanyl-(3-cyclopentyl)-L-phenylalaninate as a thick semi-solid. Ϊ H-ΝMR: (300 MHz, DMSO-dό) δ 8.18 (d, IH, J=7.8, ΝH), 7.19 (m, IH, aromatic CH), 7.13-7.00 (m, 5H, aromatic CH, ΝH), 6.77 (d, IH, J=8.8, aromatic CH), 6.63 (d, 2H, J=8.9, aromatic CH), 4.35 (m, IH, methine), 4.05 (m, IH, methine), 3.68 (s, 8H, Ν (CH 2 CH 2 C1) 2 ), 2.93 (m, 3H, ArCH 2 , cyclopentyl methine), 2.79 (m, IH, ArCH 2 ), 2.58 (m, IH, ArCH 2 ), 1.80-1.43 (m, 8H, cyclopentyl CH 2 ), 1.29 (s, 18H, t-Bu). Example 100: 4-fBisf2-chloroethvπamino -L-phenylalanyl-3-cvclopentyl-L- phenylalanine A 25 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 65 mg of tert-butyl N-((tert-butoxy)carbonyl)-4-(bis(2-chloroethyl)amino)- L-phenylalanyl-(3-cyclopentyl)-L-phenylalaninate and 8 mL of CH3ΝO 2 . The solution was acidified by bubbling HCl gas through for 10 min. After stirring at RT under N 2 for 30 min, the solution was concentrated in vacuo to dryness. Analysis of the resulting solid by HPLC (C18, 50:50:0.1 H2θ:MeCN:TFA) indicated a major component at k'=1.14 (96%) and a minor component at k'=1.60 (4%). The crude product was purified by semi-preparative HPLC (C18, 55:45:0.1→50:50:0.1 H 2 O:MeCN:TFA over 20 min, flow rate=l 5 mlJmin). Fractions containing pure product (k'=l .14 by analytical HPLC) were combined and concentrated in vacuo to 20 mL. The resulting suspension was frozen and lyophilized to afford 35 mg (55%) of 4-(bis(2-chloroethyl)amino)-L- phenylalanyl-3-cyclopentyl-L-phenylalanine as a white powder, m.p. 80-90°C. HPLC: one peak on C18, k'=1.05, 50:50:0.1 MeCN H 2 O:TFA, flow rate=l mL/min. JH-NMR: (300 MHz, DMSO-dό) δ 8.85 (d, IH, J=7.8, NH), 8.00 br s, 3H, NH3 + ), 7.25-7.00 (m, 6H, aromatic CH), 6.70 (d, 2H, J=8.5, aromatic CH), 4.50 (m, IH, methine), 3.89 (m, IH, methine), 3.69 (5, 8H, N (CH 2 CH 2 C1) 2 ), 3.12-2.83 (m, 4H, ArCH , cyclopentyl methine), 2.77 (m, IH, ArCH 2 ), 1.97 (m, 2H, cyclopentyl CH 2 ), 1.84-1.43 (m, 6H, cyclopentyl CH 2 ). 19 F-NMR: (282 MHz, DMSO-dό): d 1.26 (relative to TFA external standard). Elemental Analysis: Calcd. for C27H35N3O3C-2-TFA* 1.6 H2θ (MW 663.35): C, 52.51; H, 5.96; N, 6.33. Found: C, 52.16; H, 5.69; N, 6.69. Mass Spectrum: (FAB) 520 (M+H) + , 273. UN Spectrum: (2.5xlO" 3 M ΝaOH) λ ma χ (ε) 261.5 (18700), 303.9 (2090), λmin (ε) 232.3 (5270), 288.2 (1760). Example 101: 3-Iodo-O-ftert-butyldimethylsilvflbenzyl alcohol A 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 5.0 g of 3-iodobenzyl alcohol (Aldrich), 3.86 g of tert-butyldimethylsilyl chloride (Aldrich), 3.20 g of imidazole (Aldrich), and 30 mL of anhydrous DMF. The resulting solution was stirred under nitrogen for 4 h and concentrated in vacuo to give a clear viscous oil. This material was dissolved in 150 mL of EtOAc and the solution was washed with 5% aqueous NaHCO3 (3x70 mL), dried over anhydrous MgSO4 and concentrated in vacuo to give a clear liquid. The crude product was purified by flash chromatography on 200 g of silica gel (hexane) to afford 7.01 g (94%) of 3-iodo-O-(tert- butyldimethylsilyl)benzyl alcohol as a clear liquid. JH-NMR: (300 MHz, DMSO-dό) δ 7.67 (s, IH, aromatic CH), 7.56 (d, IH, J=7.3, aromatic CH), 7.27 (d, IH, J=9.2, aromatic CH), 7.06 (t, IH, J=7.6, aromatic CH), 4.68 (s, 2H, ArCH 2 O), 0.94 (s, 9H, t-Bu), 0.10 (s, 6H, SiMe 2 ). Example 102: 3-f 1-HvdroxycvclobutylVO-ftert-butyltrimethylsilvπbenzyl alcohol To a dry 500 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer were added 7.0 g of _3-iodo-O-(tert-butyldimethylsilyl)benzyl alcohol and 100 mL of anhydrous THF. The solution was cooled to -78°C in a dry ice-isopropanol bath and treated with 9.7 mL of 2.5 M n-butyllithium/hexane (Aldrich) by dropwise addition via syringe through a rubber septum. After stirring at -78°C for 20 min, the solution was treated with 1.80 mL of cyclobutanone (Aldrich) by dropwise addition. The reaction mixture was stirred at -78°C for 20 min and then allowed to warm to RT. After 30 min at RT the solution was mixed with lOOmL of water an subjected to rotary evaporation to remove THF. The resulting emulsion was extracted with EtOAc (4x60 mL). The combined EtOAc extracts were washed with saturated brine, dried over anhydrous MgSO4, and concentrated in vacuo to give a light yellow oil. This material was purified by flash chromatography on 160 g of silica gel (9:1 hexane:EtOAc) to afford 3.98 g (68%) of 3-(l-hydroxycyclobutyl)-O-(tert-butyltrimethylsilyl)benzyl alcohol as a clear liquid. JH-NMR: (300 MHz, DMSΟ-d6) d 7.44 (s, IH, aromatic CH), 7.38-7.23 (m, 2H, aromatic CH), 7.15 (d, IH, J=7.4, aromatic CH), 5.45 (s, IH, ΟH), 4.72 (s, 2H, ArCH 2 O), 2.41-2.18 (m, 4H, cyclobutyl CH 2 ), 1.88 (m, IH, cyclobutyl CH 2 ), 1.61 (m, IH, cyclobutyl CH 2 ), 0.90 (s, 9H, t-Bu), 0.08 (s, 6H, SiMe2). Example 103: 3-Cyclobutylbenzyl alcohol A dry 500 mL 3 -necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 3.98 g of 3-(l-hydroxycyclobutyl)-O-(tert-butyltrimethylsilyl)benzyl alcohol and 85 mL of CH 2 C1 2 . The solution was cooled to 0°C under nitrogen and treated with 15.7 mL of TFA followed by 4.78 mL of triethylsilane. After stirring at 0°C for 1 h, the solution was allowed to warm to RT. Analysis by tic (SiΟ 2 , 8:2 hexane:EtΟAc) after 3.5 h at RT indicated unreacted starting material at R =0.15 and a new component at Rf*=0.65. The solution was treated with an additional 3 mL of triethylsilane and stirred at RT for 18 h. The solution was cooled to 0°C and neutralized by slow addition of 30 mL of Et3N. The mixture was concentrated in vacuo to give a light yellow liquid which was dissolved in lOOmL of EtOAc. The resulting solution was washed with saturated brine (3x80 mL), dried over anhydrous MgSO4, and concentrated to give a clear liquid. This material was dissolved in 80 mL of anhydrous THF and treatedwith 60 mL of 1 M (n-Bu)4NF (Aldrich) in a 500 mL 3-necked flask. After stirring at RT for 18 h, the solution was concentrated in vacuo and the residue dissolved in 100 mL of EtOAc. The solution was washed with saturated brine (4x80 mL), dried over anhydrous MgSO4, and concentrated to give a clear oil. The crude product was purified by flash chromatography on 120 g of silica gel (8:2 hexane:EtOAc) to afford 1.29 g (59%) of 3-cyclobutylbenzyl alcohol as a clear liquid. JH-NMR: (300MHz, DMSO-dό) δ 7.29-7.05 (m, 4H, aromatic CH), 5.14 (t, J=5.7, OH), 4.48 (d, J=5.8, ArOfeO), 3.51 (m, IH, cyclobutyl methine), 2.30 (m, 2H, cyclobutyl CH2), 2.17-1.88 (m, 3H, cyclobutyl CH2), 1.82 (m, IH, cyclobutyl CH2). Example 104: 3 -Cvclobutylbenzyl bromide According to example 17, 1.29 g of 3-cyclobutylbenzyl alcohol was treated with 0.26 mL of PBrj in 15 mL of anhydrous CH C1 to afford 1.69 g of crude product (92% pure by 1H-NMR). This material was purified by flash chromatography on 65 g of silica gel (hexane) to afford 1.59 g (89%) of 3-cyclobutyl benzyl bromide as a clear liquid. H-NMR: (300 MHz, DMSO-dό) δ 7.35-7.13 (m, 4H, aromatic CH), 4.70 (s, 2H, ArCH2Br), 3.53 (m, IH, cyclobutyl methine), 2.39-1.75 (m, 6H, cyclobutyl CH2). Example 105: tert-Butyl f2R. 3S. 5_SV6-oxo-2.3-diphenyl-5-f3-cvclobutylbenzvn-4- moφholinecarboxylate According to example 81, 2.50 g of tert-butyl (2R, 3S)-(-)-6-oxo-2,3-diphenyl-4- moφholinecarboxylate (Aldrich) was alkylated with 1.59 g of 3-cyclobutylbenzyl bromide in 80 mL of anhydrous THF using 7.41 mL of 1 M sodium bis(trimethylsilyl)amide in THF (Aldrich). The crude product was subjected to flash chromatography on 120 g of silica gel (8:2 hexane:EtOAc) and then recrystallization from EtOH-water to afford 1.90 g (54%) of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diρhenyl- 5-(3-cyclobutylbenzyl)-4-moφholinecarboxylate as a white powder, m.p. 145-147°C. Example 106: f2R. 3S. 5__A-6-Oxo-2.3-diphenyl-5-f3-cvclobutylbenzyl ' .moφholine According to example 82, 1.87 g of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3- cyclobutylbenzyl)-4-moφholinecarboxylate was treated with 3.1 mL of TFA in 30 mL of CH 2 C_2 for 4 h. Neutralization with 8 mL of Et3N and work-up in the usual manner gave a light yellow residue. Purification of the crude material by flash chromatography on 85 g of silica gel (8:2 hexane:EtOAc) afforded 1.35 g (91%) of (2R, 3S, 5S)-6-oxo- 2,3-diphenyl-5-(3-cyclobutylbenzyl)moφholine as a thick, transparent semi-solid. -1H-NMR: (300 MHz, DMSO-dό) δ 7.24-7.02 (m, 10H, aromatic CH), 6.92 (m, 4H, aromatic CH), 5.71 (s, IH, moφholine 2-H), 4.64 (m, IH, moφholine 3-H), 4.24 (m, IH, moφholine 5-H), 3.42 (m, IH, cyclobutyl methine), 3.26 (m, IH, ArCH2), 3.10 (m, IH, A1-CH2), 2.76 (t, IH, J=5.3, NH), 2.15 (m, 2H, cyclobutyl CH2), 2.02-2.81 (m, 3H, cyclobutyl CH2), 1.71 (m, IH, cyclobutyl CH2). Example 107: 3-Cyclobutyl-L-phenylalanine According to example 53, 1.35 g of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3- cyclobutylbenzyl)moφholine was hydrogenolyzed in 60 mL of 1 : 1 THF:EtOH using 0.42 g of PdCl2 to afford 0.79 g (91%) of 3-cyclobutyl-L-phenylalanine HCl as a white powder, m.p. >200°C. JH-NMR: (300 MHz, DMSO-dό) δ 8.40(br s, 3H, NH3 + ), 7.26 (m, IH, aromatic CH), 7.13 (m, 2H, aromatic CH), 7.08 (d, IH, J=7.4, aromatic CH), 4.13 (m, IH, methine), 3.50 (m, IH, cyclobutyl methine), 3.11 (d, 2H, J=7.4, ArCH2), 2.29 (m, 2H, cyclobutyl CH2), 2.18-1.88 (m, 3H, cyclobutyl CH2), 1.83 (m, IH, cyclobutyl CH2). Example 108: tert-Butyl 3-cyclobutyl-L-phenylalaninate According to example 54, 0.65 g of 3-cyclobutyl-L-phenylalanine HCl was treated with 25 mL of isobutylene in 25 mL of 1,4-dioxane in the presence of 0.5 mL of cone H2SO4. Work-up in the usual manner followed by acidification of the free amine with ethereal HCl afforded 0.64 g (81%) of tert-butyl 3-cyclobutyl-L-phenylalaninate HCl as a white powder, m.p. 173-174°C. Example 109: tert-Butyl N-ffbenzyloxy ' .carbonyl ' .-5-O-tert-butyl-L-glutam-l-yl-3- cvclobutyl-L-phenylalaninate According to example 9, 0.64 g of tert-butyl 3-cyclobutyl-L-phenylalaninateΗCl was coupled with 0.69 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.41 g of EDC (Aldrich) and 0.23 mL of Νrmethylmoφholine. The crude product was purified by flash chromatography on 85 g of silica gel (7:3 hexane.EtOAc) to afford 0.98 g (80%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-l-yl-3-cyclobutyl-L- phenylalaninate as a thick transparent oil. *H-ΝMR: (300 MHz, DMSO-dό) δ 8.21 (d, IH, J=7.3, ΝH), 7.33 (m, 6H, aromatic CH, ΝH), 7.18 (m, IH, aromatic CH), 7.02 (m, 3H, aromatic CH), 5.00 (m, 2H, PhCH2θ), 4.32 (m, IH, methine), 4.05 (m, IH, methine), 3.47 (m, IH, cyclobutyl methine), 2.92 (d, 2H, J=7.6, ArCH 2 ), 2.31-2.12 (m, 4H, glu 4-CH 2 , cyclobutyl CH 2 ), 2.11-1.62 (m, 6H, glu 3-CH 2 , cyclobutyl CH2), 1.37 (s, 9H, t-Bu), 1.29 (s, 9H, t-Bu). Example 110: tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-3-cvclobutyl-L-phenylalaninate According to example 48, 0.98 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L- glutam-l-yl-3-cyclobutyl-L-phenylalaninate was hydrogenolyzed using 0.17 g of 10% Pd(C) in 55 mL of MeOH. This afforded 0.76 g (100%) of tert-butyl 5-O-tert-butyl-L- glutam-l-yl-3-cyclobutyl-L-phenylalaninate as a thick transparent oil. A sample of the corresponding HCl salt for ^H-ΝMR analysis was prepared by treating 10 mg of the product with excess IN ethereal HCl and concentrating to dryness. *H-NMR: (300 MHz, DMSO-d6) δ 8.92 (d, IH, J=7.0, NH), 8.25 (br s, 3H, NH3 + ), 7.20 (m, IH, aromatic CH), 7.08 (m, 3H, aromatic CH), 4.38 (m, IH, methine), 3.83 (m, IH, methine), 3.48 (m, IH, cyclobutyl methine), 2.94 (d, 2H, J=7.8, ArCH2), 2.16 (m, 4H, glu 4-CH2, cyclobutyl CH2), 2.15-1.86 (m, 5H, glu 3-CH2, cyclobutyl CH2), 1.79 (m, IH, cyclobutyl CH2), 1.38 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu). Example 111: tert-Butyl N-f4-f f2.4-diamino-6- pteridinvπmethvπmethylamino'.benzoylV5-O-tert-butyl-L-glutam-l-yl-3-cyclobutyl-L- phenylalaninate According to example 49, 0.285 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.38 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3-cyclobutyl-L-phenylalaninate using 0.34 mL of DECP (Aldrich) and 0.37 mL of Et3Ν in 25 mL of DMF. The crude product was subjected to flash chromatography on 100 g of silica gel (7:7:1 CH2Cl2.acetone:MeΟH) to afford 0.45 g (78%) of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam- 1 -yl-3 -cyclobutyl-L- phenylalaninate as a yellow powder, m.p. 120-125°C. Example 112: N-f4-fff2.4-Diamino-6-pteridinyl ' .methvnmethylamino'.benzoylVL- glutam- 1 -yl-3-cyclobutyl-L-phenylalanine According to example 50, 0.40 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3-cyclobutyl-L- phenylalaninate was treated with gaseous HCl in 60 mL of CH3ΝO2 for 1 h. Work-up and isolation in the usual manner afforded 0.281 g (79%) of N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l -yl-3-cyclobutyl-L-phenylalanine as a yellow powder, m.p. 175°C (dec). HPLC: one peak on C18, k'=2.50, 65:35:0.1 H2θ:MeCN:TFA, flow rate=l mL/min. JH-NMR: (300 MHz, DMSO-dό) δ 12.85-11.75 (br m, COOH), 8.58 (s, IH, pteridinyl 7-CH), 8.04 (d, IH, J=8.1, amide NH), 8.00 (d, IH, J=8.7, amide NH), 7.75 (br s, IH, NH2), 7.71 (d, 2H, J=8.9, aromatic CH), 7.55 (br s, IH, NH2), 7.10-6.92 (m, 4H, aromatic CH), 6.82 (d, 2H, J=8.9, aromatic CH), 6.70 (br s, 2H, NH2), 4.79 (s, 2H, pteridinyl-CH2), 4.39 (m, 2H, methines), 3.40 (m, IH, cyclobutyl methine), 3.21 (s, 3H, N-CH3), 3 -00 (m, IH, ArCH2), 2.88 (m, IH, ArCH2), 2.29-2.08 (m, 4H, glu 4-CH2, cyclobutyl CH2), 2.07-1.65 (m, 6H, glu 3-CH2, cyclobutyl CH2). Elemental Analysis: Calcd. for C33H37N9O6 I 6 H2O (MW 684.54): C, 57.90; H, 5.92; N, 18.42. Found: C, 57.88; H, 5.90; N, 18.52. Mass Spectrum: (Ion Spray) 656 (M+H) + . UN Spectrum: (pH 7 Buffer) λ max (ε) 259.5(22100), 308.0 (22300), 373.7 (6740). λ min (ε) 241.2 (12900), 274.5 (14600), 345.6 (5130). Example 113: 6-Bromomethyl-3.4-dihvdro-2-methyl-4-oxoquinazoline According to example 4, 1.00 g of 3,4-dihydro-2,6-dimethyl-4-oxoquinazoline (Sen, A. B.; Gupta, J. K. J. Indian Chem. Soc, 1962, 31, 369) was subjected to bromination using 1.05 g of N-bromosuccinimide (Aldrich) and 0.17 g of benzoyl peroxide in 50 mL of 1,2-dichloro-ethane for 30 min to afford 0.95 g (64%) of 6-bromomethyl-3,4-dihydro- 2-methyl-4-oxoquinazoline as a tan solid. ^-ΝMR: (300 MHz, DMSO-dό) δ 8.17 (s, IH, aromatic CH), 7.85 (d, IH, J=8.5, aromatic CH), 7.58 (d, IH, J=8.5, aromatic CH), 4.88 (s, 2H, ArCH2Br), 2.39 (s, 3H, CH3). Example 114: Ethyl 5-N-methylamino-2-thiophenecarboxylate A mixture of 2.0 g of ethyl 5-amino-2-thiophenecarboxylate (Mackay, D. Can. J. Chem., 1966, 44, 2881-2891; Paul, H.; Migulla, H. Arch. Pharm., 1978, 311, 679- 691.), 2.05 g of methyl iodide (Aldrich), and 2.58 g of 2,6-lutidine (Aldrich) in 80 mL of DMF was stirred at 70°C under nitrogen for 24h. Analysis of the reaction mixture by tic (SiO 2 , 3:2 hexane:EtOAc) indicated starting material at Rf=0.51 and a new componenent at R =0.60. The mixture was treated with an additional 1.71 g of methyl iodide and was stirred at 70°C for 8h, RT for 72h and 70°C for an additional 8h. The reaction mixture was mixed with 75 mL of water and extracted with EtOAc (3x150 mL). The combined EtOAc extracts were washed with saturated aqueous brine, dried over anhydrous Νa2Sθ4 and concentrated in vacuo to dryness. The crude residue was subjected to flash chromatography on silica gel (75:25 hexane:EtOAc) to afford 0.67 g (38%) of ethyl 5-N-methylamino-2-thiophenecarboxylate as a brown solid, m.p. 41- 43°C. Example 115: Ethyl 5-fff3.4-dihvdro-2-methyl-4-oxo-6- quinazolinyl methvDmethylaminoV2-thiophene-carboxylate A 100 mL round bottomed flask equipped with a condenser and a magnetic stirrer was charged with 0.66 g of 6-bromomethyl-3,4-dihydro-2-methyl-4-oxoquinazoline, 0.44 g of ethyl 5-N-methylamino-2-thiophenecarboxylate, 0.25 g of 2,6-lutidine, and 30 mL of anhydrous DMF. The mixture was stirred at 80°C for 18h. Analysis of the reaction mixture by tic (Siθ2, 9 5:5 CH2Cl2:MeOH) indicated starting materials and a new component at Rf=0.41. The mixture was concentrated in vacuo to dryness and the residue was subjected to flash chromatography on 110 g of silica gel (95:5 CH 2 Cl2.MeOH). Fractions containing the Rf=0.41 component were combined and concentrated to afford 0.42 g (49%) of ethyl 5-(((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)methylamino)-2-thiophenecarboxylate as a light tan powder, m.p. >200°C. -iH-NMR: (300 MHz, DMSO-dό) δ 12.22 (br s, IH, NH), 7.92 (s, IH, aromatic CH), 7.65 (d, IH, J=8.4, aromatic CH), 7.56 (d, IH, J=8.5, aromatic CH), 7.48 (d, IH, J=4.4, thienyl CH), 6.07 (d, IH, J=4.3, thienyl CH), 4.70 (s, 2H, quinazolinyl-CH2), 4.16 (q, 2H, J=7.2, ethyl CH2), 3.09 (s, 3H, N-CH3), 2.33 (s, 3H, quinazolinyl 2-CH3), 1.22 (t, 3H, J=7.2, ethyl CH3). Example 116: 5-fff3.4-Dihvdro-2-methyl*-4-oxo-6-quinazolinv0methvDmethylaminoV2- thiophene-carboxylic acid A 25 mL 3-necked flask equipped with a condenser, a thermometer, and a magnetic stirrer was charged with 0.20 g of ethyl 5-(((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)methylamino)-2-thiophene-carboxylate, 6 mL of EtOH, and 6 mL of IN aqueous NaOH. The resulting solution was stirred under nitrogen at RT for 25 min and then at 55°C for 2h. Analysis by tic (Siθ2, 95:5 CH2CI2) indicated no remaining starting material and a new component at Rf=0.06. The solution was cooled to RT and subjected to rotary evaporation to remove EtOH. The remaining solution was diluted with 10 mL of water and acidified to pH=3.5 by addition of IN aqueous HCl. A tan precipitate formed which was collected by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 0.116 g (63%) of 5-(((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thiophene- carboxylic acid as a white powder, m.p. 195°C (dec). iH-NMR: (200 MHz, DMSO-dό) δ 12.40-11.90 (br m, COOH, NH), 7.95 (s, IH, aromatic CH), 7.69 (m, IH, aromatic CH), 7.58 (d, IH, J=8.2, aromatic CH), 7.42 (d, IH, J=4.2, thienyl CH), 6.04 (d, IH, J=4.2, thienyl CH), 4.70 (s, 2H, quinazolinyl-CH2), 3.09 (s, 3H, N-CH3), 2.35 (s, 3H, quinazolinyl 2-CH3). Example 117: tert-Butyl N-ff5-f(f 3.4-dihvdro-2-methyl-4-oxo-6- quinazolinyπmethyπmethylanιinoV2-thienvπcarbonyl'.-5-O-tert-butyl-L-glutam-l-yl-3- cyclopentyl-L-phenylalaninate A dry 50 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 0.115 g of 5-(((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)methylamino)-2-thiophene-carboxylic acid, 0.165 g of tert-butyl 5- O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate 0.06 mL of Et3Ν, and 7 mL of anhydrous DMF. The yellow solution was treated with 0.06 mL of DECP and was stirred at RT under nitrogen. After 2h, tic (Siθ2, 95:5 CH2C_2:MeΟH) indicated starting material at Rf=0.02, a major new component at Rf=0.37, and minor components at Rf=0.31 and 0.40. The reaction mixture was treated with an additional 0.03 mL of DECP and 0.03 mL of Et3N and was stirred for another lh at RT. TLC indicated little additional conversion. The solution was concentrated in vacuo to dryness and the residue dissolved in 60 mL of CHCI3. The solution was washed with 5% aqueous NaHCθ3 (3x50 mL), dried over anhydrous MgSθ4, and concentrated to give a light yellow oil. The crude product was subjected to flash chromatography twice (Siθ2, 95:5 EtOAcMeOH; Siθ2, 97:3 CH2Cl2:MeOH) to afford 0.119 g (46%) of tert-butyl N-((5- (((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)methylamino)-2-thienyl)carbonyl)- 5-O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate as a tan foam. JH-ΝMR: (400 MHz, DMSO-dό) δ 12.19 (s, IH, quinazoline ΝH), 8.18 (d, IH, J=7.1, amide ΝH), 7.92 (m, 2H, amide ΝH, aromatic CH), 7.63 (dd; IH; J=7.9,1.8; aromatic CH), 7.57 (d, IH, J=4.1, thienyl CH), 7.54 (d, IH, J=8.0, aromatic CH), 7.12-6.94 (m, 4H, aromatic CH), 5.97 (d, IH, J=4.1, thienyl CH), 4.63 (s, 2H, quinazolinyl-CH2), 4.40-4.27 (m, 2H, methines), 3.02 (s, 3H, Ν-CH3), 2.87 (m, 3H, ArCH2, cyclopentyl methine), 2.31 (s, 3H, quinazolinyl 2-CH3), 2.20 (t, 2H, J=7.7, glu 4-CH2), 1.91 (m, 3H, glu 3-CH2, cyclopentyl CH2), 1.83-1.40 (m, 7H, cyclopentyl CH2), 1.35 (s, 9H, t-Bu), 1.27 (s, 9H, t-Bu). Example 118: N-ff5-fff3.4-Dihvdro-2-methyl-4-oxo-6- quinazolinyl ' .methyl')methylamino'.-2-thienyl ' .carbonyl ' )-L-glutam-l-yl-3-cvclopentyl-L- phenylalanine According to example 50, 0.117 g of tert-butyl N-((5-(((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)methylamino)-2-thienyl)carbonyl)-5-O-tert-butyl-L-glutam-l-yl-3- cyclopentyl-L-phenylalaninate was treated with gaseous HCl in 25 mL of CH3ΝO2 for 45 min. Removal of the CH3NO2 in vacuo gave a tan residue which was purified by semi-preparative reverse phase HPLC (C18, 70 : 30 : 0.1 → 60 : 40 : 0.1 H2O : MeCN : TFA over 25min ). Fractions containing the major component (k'=1.61 on analytical HPLC, C18, 60:40:0.1 H2θ:MeCN:TFA) were combined, concentrated to 25 mL and lyophilized to afford 0.052 g (42%) of N-((5-(((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)methylamino)-2-thienyl)carbonyl)-L-glutam-l-yl-3-cyclopentyl-L- phenylalanine as a yellow powder, m.p. 125°C (dec). HPLC: one peak on C18, k'=1.62, 60:40:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. *H-NMR: (300 MHz, DMSO-dό) δ 8.09 (d, IH, J=7.5, amide NH), 7.93 (m, 2H, amide NH, aromatic CH), 7.69 (d, IH, J=8.0, aromatic CH), 7.56 (m, 2H, aromatic CH, thienyl CH), 7.10-6.92 (m, 4H, aromatic CH), 5.97 (d, IH, J=4.3, thienyl CH), 4.66 (s, 2H, quinazolinyl-CH2), 4.33 (m, 2H, methines), 3.06 (s, 3H, N-CH3), 2.99 (m, IH, ArCH2), 2.85 (m, IH, ArCH2), 2.38 (s, 3H, quinazolinyl 2-CH3), 2.22 (t, 2H, J=7.5, glu 4-CH2), 2.02-1.37 (m, 10H, glu 3-CH2, cyclopentyl CH2). 19 F-NMR: (376 MHz, DMSO-dό) δ 0.57 (relative to TFA external standard). Elemental Analysis: Calcd. for C35H39N5O7S I.2 TFA- 1.3 H2O (MW 834.03): C, 53.86; H, 5.17; N, 8.40; S, 3.84. Found: C, 53.85; H, 5.11; N, 8.36; S, 3.92. Mass Spectrum: (Ion Spray) 691 (M+NH4 + ). UN Spectrum: (pH 7 Buffer) λ max (ε) 225.2 (36600), 265.6 (10300), 353.1 (22200). λmin (ε) 214.3 (35200), 252.1 (9230), 288.4 (4120). Example 119: tert-Butyl 5-O-tert-butyl-N-f4-ff3-f2.4-diamino-1.6-dihvdro-6-oxo-5- p\rimidinyl ' .propylVtrifluoroacetamido ' .benzoylVL-glutam-l-yl-3-cvclopentyl-L- phenylalaninate A 25 mL 3 -necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.258 g of 4-(N-(3-(2,4-diamino-l,6-dihydro-6-oxo-5- ρyrimidinyl)propyl)trifluoroacetamido)benzoic acid (Styles, N. L., et al, J. Heterocvclic Chem. . 1990, 27, 1809), 0.250 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3- cyclopentyl-L-phenylalaninate, 0.077 g of 1-hydroxybenzotriazole hydrate (Aldrich), 5 mL of anhydrous DMF, and 0.07 mL of Et3Ν. The solution was treated with 0.104 g of DCC (Fluka) and stirred at RT under nitrogen for 24h. The mixture was filtered and the filtrate was concentrated in vacuo to dryness. The residue was dissolved in 70 mL of CH2CI2, washed with 5% aqueous NaHCO3 (3x50 mL), dried over anhydrous MgSO4 and concentrated to give a tan oil. This material was purified by flash chromatography on silica gel (95:5 _4__ 90:10 CH2Cl2:MeOH) to afford 0.304 g (70%) of tert-butyl 5-O- tert-butyl-N-(4-((3-(2,4-diamino- 1 ,6-dihydro-6-oxo-5- pyrirnidinyl)propyl)trifluoroacetamido)-benzoyl)-L-glutam- 1 -yl-3 -cyclopentyl-L- phenylalaninate as a tan glass, m.p. 124-130°C. Example 120: tert-Butyl 5-O-tert-butyl-N-f4-ff3-f2.4-diamino-1.6-dihvdro-6-oxo-5- pyrimidinyl ' .propyπamino')benzoyl ' .-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate To a 100 mL round bottomed flask equipped with a nitrogen inlet and a magnetic stirrer were added 0.268 g of tert-butyl 5-O-tert-butyl-N-(4-((3-(2,4-diamino-l,6-dihydro-6- oxo-5-pyrimidinyl)propyl)trifluoroacetamido)benzoyl)-L-glutam-l-yl-3-cyclopentyl-L- phenylalaninate, and 7 mL of MeOH. The solution was treated with 0.18 mL of 40% (w/w) dimethylamine/water (Aldrich) followed by 0.45 mL of water and was stirred at RT under nitrogen for 24h. The solution was concentrated in vacuo to dryness and the residue was subjected to flash chromatography on 60 g of silica gel (92:8 CH 2 Cl :MeOH) to afford 0.185 g (78%) of tert-butyl 5-O-tert-butyl-N-(4-((3-(2,4- dian_ino-l,6-dihydro-6-oxo-5-pyriπύdinyl)propyl)amino)benzoyl)-L-glutam-l-yl-3- cyclopentyl-L-phenylalaninate as a thick transparent semi-solid. H-ΝMR: (200 MHz, DMSO-dό) δ 9.80 (br s, IH, pyrimidinyl NH), 8.20 (d, IH, J=7.2, amide NH), 7.90 (d, IH, J=8.0, amide NH), 7.67 (d, 2H, J=8.6, aromatic CH), 7.20-6.98 (m, 4H, aromatic CH), 6.55 (d, 2H, J=8.6, aromatic CH), 6.25 (m, IH, NH), 5.94 (br s, 2H, NH 2 ), 5.77 (br s, 2H, NH2), 4.54-4.29 (m, 2H, methines), 3.12-2.80 (m, 5H, cyclopentyl methine, ArCH2, pyrimidinyl-CH 2 CH 2 CH2N), 2.27 (m, 4Η, glu 4-CH 2 , pyrimidinyl-CH2CH 2 CH 2 N), 2.10-1.82 (4H, glu 3-CH 2 , cyclopentyl CH 2 ), 1.80-1.45 (m, 8H, cyclopentyl CH2, pyrimidinyl-CH2CH2CH 2 N), 1.39 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu). Example 121: N-f4-ff3-f2.4-Diamino-1.6-dihvdro-6-oxo-5- pyrimidinyl'.propyπarnino > .benzoylVL-glutam-l-yl-3-cyclopentyl-L-phenylalanine A 100 mL round bottomed flask equipped with a magnetic stirrer was charged with 0.180 g of tert-butyl 5-O-tert-butyl-N-(4-((3-(2,4-diamino-l,6-dihydro-6-oxo-5- pyrimidinyl)propyl)amino)benzoyl)-L-glutam- 1 -yl-3-cyclopentyl-L-phenylalaninate and 25 mL of CH3ΝO2. The mixture was acidified by bubbling HCl gas through for 5 min, however, the solid starting material did not dissolve. Analysis of the reaction mixture by tic (Siθ2, 9 2:8 CH2C_2:MeOH) indicated minimal conversion of the starting material. The mixture was concentrated in vacuo to dryness. A yellow residue resulted which was suspended in 20 mL of CH2CI2 and treated with 1 mL of anisole followed by 15 mL of TFA. The solid quickly dissolved giving a yellow solution which was stirred at RT under nitrogen. After lh, tic indicated no remaining starting material and a new spot at R O.O. The solution was concentrated to dryness and the residue was purified by semi- preparative reverse phase HPLC (C18, 70:30:0.1 H2O: MeCN:TFA). Fractions containing pure material were combined, concentrated to 30 mL and lyophilized to afford 0.098 g (47%) ofN-(4-((3-(2,4-diamino-l,6-dihydro-6-oxo-5- pyrimidinyl)propyl)amino)benzoyl)-L-glutam- 1 -yl-3-cyclopentyl-L-phenylalanine as a fluffy white solid, m.p. 125°C (dec). HPLC: one peak on C18, k'=1.64, 70:30:0.1 H2θ:MeCΝ:TFA, flow rate=lmL/min. -1H-NMR: (300 MHz, DMSO-dό) δ 12.91-11.10 (br s, COOH, pyrimidinyl NH), 8.07 (d, IH, J=7.6, amide NH), 7.88 (d, IH, J=7.9, amide NH), 7.70 (br s, IH, NH2), 7.62 (m, 3H, NH2, aromatic CH), 7.13-6.70 (m, 6H, NH2, aromatic CH), 6.53 (d, 2H, J=8.6, aromatic CH), 4.40 (m, 2H, methines), 3.01 (m, 3H, cyclopentyl methine, pyrimidinyl- CH2CH2CH2N), 2.86 (m, 2Η, ArCH2), 2.25 (m, 4H, glu 4-CH2, pyrimidinyl- CH2CH2CH2N), 2.01-1.78 (m, 4H, glu 3-CH2, cyclopentyl CH2), 1.76-1.37 (m, 8H, cyclopentyl CH2, pyrimidinyl-CH2CH2CH2N). 19 F-NMR: (282 MHz, DMSO-dό) δ 3.92 (relative to TFA external standard). Elemental Analysis: Calcd. for C33H41N7O7 I.8 TFA- 1.6 H2O (MW 881.80): C, 49.85; H, 5.26; N, 11.12. Found: C, 49.81; H, 5.24; N, 11.11. Mass Spectrum: (Ion Spray) 648 (M+H) + . UN Spectrum: (pH 7 Buffer) λ ma χ (ε) 280.1 (29700). λmin (ε) 247.4 (7590). sh (e) 302.9 (20400). Example 122: Di-tert-butyl N-ffbenzyloxy'.carbonylV5-O-tert-butyl-L-glutam-l-yl-L- aspartate According to example 9, 1.00 g of L-aspartic acid di-t-butyl ester hydrochloride (Sigma) was coupled with 1.20 g of N-Cbz-L-glutamic acid γ-t-butyl ester (Sigma) using 0.72 g of EDC (Aldrich) and 0.39 mL of N-methylmoφholine. The crude product was purified by flash chromatography on 80 g of silica gel (75:25 hexane:EtOAc) to afford 1.10 g (55%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-l-yl-4-O-tert- butyl-L-aspartate as a white solid, m.p. 119-120°C. Example 123: Di-tert-butyl 5-O-tert-butyl-L-glutam-l-yl-L-aspartate According to example 48, 0.389 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl- L-glutam-l-yl-4-O-tert-butyl-L-aspartate was hydrogenolyzed using 0.07 g of 10% Pd(C) in 40 mL of MeOH. This afforded 0.29 g (98%) of di-tert-butyl 5-O-tert-butyl-L- glutam-1-yl-L-aspartate as a thick transparent oil. A sample of the corresponding HCl salt for iH-ΝMR analysis was prepared by treating 10 mg of the product with excess IN ethereal HCl and concentrating to dryness. *H-NMR: (300 MHz, DMSO-dό) δ 8.91 (d, IH, J=7.8, NH), 8.31 (br s, 3H, NH3+), 4.52 (m, IH, methine), 3.83 (m, IH, methine), 2.63 (d, 2H, J=6.1, asp CH2), 2.36 (m, 2H, glu 4-CH 2 ), 1.94 (m, 2H, glu 3-CH 2 ), 1.38 (s, 27H, t-Bu). Example 124: Di-tert-butyl 5-O-tert-butyl-N-f4-fff2.4-diamino- ό-pteridinyπmethyD methylamino'.benzoyl'.-L-glutam- 1 -yl-L-aspartate According to example 49, 0.30 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.34 g of di-tert-butyl 5-O-tert-butyl-L-glutam-l-yl-L-aspartate using 0.36 mL of DECP (Aldrich) and 0.39 mL of Et3Ν in 25 mL of DMF. The crude product was subjected to flash chromatography twice (Siθ2,7:7:l CH2C_2:acetone:MeOH; Siθ2, 96:4 - 95:5 CH2Cl2:MeOH) to afford 0.213 g (37%) of di-tert-butyl 5-O-tert-butyl- N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-L- aspartate as a yellow powder, m.p. 130-135°C. Example 125 : N- 4-f f f 2.4-Diamino-6-pteridinyl ' .methyl.methylamino ' .benzoyn-L- glutam-1-yl-L-aspartic acid (Procedure B) For Procedure A to prepare this compound, see Example 33. According to example 50, 0.307 g of di-tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino- 6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-L-aspartate was treated with gaseous HCl in 40 mL of CH3ΝΟ 2 for 1.5h. The reaction mixture was concentrated to 5 mL and mixed with 50 mL of ether. The resulting solid was collected by filtration and dried in vacuo. The product was dissolved in 25 mL of water, filtered and lyophilized to afford 0.246 g (89%) of N* 4-(((2,4-di__mino-6-pteridinyl)methyl)methylamino)benzoyl)- L-glutam-1-yl-L-aspartic acid as a yellow-orange powder, m.p. 160°C (dec). HPLC: one peak on C18, k'=0.70, 85:15:0.1 H 2 Ο:MeCΝ:TFA, flow rate=l mL/min. *H-NMR: (300 MHz, DMSO-dό) δ 13.12 (br s, COOH), 9.31 (s, IH, NH 2 ), 9.08 (s, IH, NH 2 ), 8.72 (s, IH, pteridinyl 7-CH), 8.68 (br s, IH, NH 2 ), 8.22 (d, IH, J=8.0, amide NH), 8.09 (d, IH, J=7.9, amide NH), 7.79 (br s, IH, NH 2 ), 7.75 (d, 2H, J=8.9, aromatic CH), 6.82 (d, 2H, J=8.9, aromatic CH), 4.87 (s, 2H, pteridinyl-CH2), 4.59-4.40 (m, 2H, methines), 3.24 (s, 3H, N-CH3), 2.75-2.52 (m, 2H, asp CH2), 2.27 (t, 2H, J=7.8, glu 4-CH2), 2.10-1.78 (m, 2H, glu 3-CH 2 ) . Elemental Analysis: Calcd. for C24H27N9O8 2 HCl* 1.3 H 2 O (MW 665.87): C, 43.29; H, 4.78; N, 18.93; CI, 10.65. Found: C, 43.42; H, 4.90; N, 18.83; CI, 10.46. Mass Spectrum: (FAB) 570 (M+H) + . UN Spectrum: (pH 7 Buffer) λ m ax (ε) 258.7 (21900), 306.4 (23800), 372.2 (7250). λmin (ε) 240.1 (13400), 272.4 (15200), 345.0 (5740). Example 126: Di-tert-butyl N-(fbenzyloxy , .carbonylV5-O-tert-butyl-L-glutam-l-yl-L- glutamate According to example 9, 0.88 g of L-glutamic acid di-t-butyl ester hydrochloride (Sigma) was coupled with 1.00 g of N-Cbz-L-glutamic acid γ-t-butyl ester (Sigma) using 0.60 g of EDC (Aldrich) and 0.33 mL of N-methylmoφholine. The crude product was purified by flash chromatography on silica gel (75:25 hexane:EtOAc) to afford 1.49 g (87%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam- 1 -yl-5-O-tert-butyl-L- glutamate as a thick transparent oil. ^-ΝMR: (300 MHz, DMSO-dό) δ 8.21 (d, IH, J=7.6, ΝH), 7.42 (d, IH, J=8.2, ΝH), 7.33 (m, 5H, aromatic CH), 5.01 (m, 2H, PhCH2θ), 4.17-3.96 (m, 2H, methines), 2.24 (m, 4H, glu 4-CH2), 1.96-1.64 (m, 4H, glu 3-CH2), 1.37 (s, 27H, t-Bu). Example 127: Di-tert-butyl 5-O-tert-butyl-L-glutam-l-yl-L-glutamate According to example 48, 0.60 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L- glutam-l-yl-5-O-tert-butyl-L-glutamate was hydrogenolyzed using 0.10 g of 10% Pd(C) in 45 mL of MeOH. This afforded 0.45 g (98%) of di-tert-butyl 5-O-tert-butyl-L- glutam-1-yl-L-glutamate as a thick transparent oil. A sample of the corresponding HCl salt for 1H-ΝMR analysis was prepared by treating 10 mg of the product with excess IN ethereal HCl and concentrating to dryness. ^-NMR: (200 MHz, DMSO-dό) δ 8.86 (d, IH, J=7.3, NH), 8.31 (br s, 3H, NH3 + ), 4.21 (m, IH, methine), 3.87 (m, IH, methine), 2.35 (m, 4H, glu 4-CH2), 2.07-1.66 (m, 4H, glu 3-CH2), 1.39 (s, 27H, t-Bu). Example 128: Di-tert-butyl 5-O-tert-butyl-N-f4-fff2.4-diamino- 6-pteridinyl_methyl.methylamino ' .benzoylVL-glutam- 1 -yl-L-glutamate According to example 49, 0.30 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.386 g of di-tert-butyl 5-O-tert-butyl-L-glutam-l-yl-L-glutamate using 0.36 mL of DECP (Aldrich) and 0.39 mL of Et3Ν in 25 mL of DMF. The crude product was subjected to flash chromatography on 150 g of silica gel (7:7:1 CH2Cl2:acetone:MeOH) to afford 0.432 g (73%) of di-tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino- 6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-L-glutamate as a yellow powder, m.p. 130-145°C. Example 129: N-(4-fff2.4-Diamino-6-pteridinyl ' .methvDmethylamino)benzoyl'.-L- glutam-1-yl-L-glutamic acid fProcedure B) For Procedure A to prepare this compound, see Example 39. According to example 50, 0.382 g of di-tert-butyl 5-O-tert-butyl-N-(4-(((2,4-diamino- 6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-L-glutamate was treated with gaseous HCl in 50 mL of CH3ΝΟ2 for lh. The reaction mixture was concentrated to 10 mL and mixed with 50 mL of ether. The resulting solid was collected by filtration and dried in vacuo. The product was dissolved in 25 mL of water, filtered and lyophilized to afford 0.265 g (76%) of N-(4-(((2,4-diaπ_ino-6-pteridinyl)methyl)methylamino)benzoyl)- L-glutam-1-yl-L-glutamic acid as a yellow-orange powder, m.p. 155°C (dec). HPLC: one peak on C18, k'=0.88, 85:15:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. -1H-NMR: (300 MHz, DMSO-dό) δ 13.18 (br s, COOH), 9.29 (s, IH, NH2), 9.07 (s, IH, NH2), 8.72 (s, IH, pteridinyl 7-CH), 8.69 (br s, IH, NH2), 8.19 (d, IH, J=7.7, amide NH), 8.09 (d, IH, J=7.9, amide NH), 7.87 (br s, IH, NH2), 7.74 (d, 2H, J=8.9, aromatic CH), 6.81 (d, 2H, J=8.9, aromatic CH), 4.87 (s, 2H, pteridinyl-CH2), 4.42 (m, IH, methine), 4.19 (m, IH, methine), 3.24 (s, 3H, N-CH3), 2.28 (m, 4H, glu 4-CH2), 2.07-1.71 (m, 4H, glu 3-CH2). Elemental Analysis: Calcd. for C25H29 9O8 I.9 HCl 2 H2O (MW 688.87): C, 43.59; H, 5.11; N, 18.30; CI, 9.78. Found: C, 43.72; H, 5.07; N, 18.16; CI, 9.97. Mass Spectrum: (FAB) 584 (M+H) + . UN Spectrum: (pH 7 Buffer) λ ma χ (ε) 258.8(23700), 306.2 (25600), 371.9 (7830). λmin (ε) 240.4 (14300), 272.9 (16600), 344.6 (6150). Example 130: N-f4-f( f 2.4-Diamino-6-pteridinyl)methyl > .methylamino.benzoylVL- glutam-l-yl-3-tert-butyl-L-phenylalanine fProcedure B^ For Procedure A to prepare this compound, see Example 97. According to example 50, 3.51 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methyl__mino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3-tert-butyl-L- phenylalaninate was treated with gaseous HCl in 150 mL of CH3ΝΟ2 for lh. The reaction mixture was concentrated in vacuo to a slurry and mixed with 100 mL of ether. The solid was collected by vacuum filtration and dissolved in 150 mL of 6:4 MeCN:H2θ. The solution was subjected to rotary evaporation to remove MeCN and the resulting suspension was frozen and lyophilized to afford 3.167 g (93%) of N-(4- (((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glut am- 1 -yl-3 -tert-butyl-L- phenylalanine as a yellow-orange powder, m.p. 165°C (dec). HPLC: one peak on C18, k'=2.30, 70:30:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. ^-NMR: (300 MHz, DMSO-dό) δ 13.35-12.95 (br s, COOH), 9.30 (s, IH, NH2), 9.07 (s, IH, NH2), 8.71 (s, IH, pteridinyl 7-CH), 8.68 (br s, IH, NH2), 8.14 (d, IH, J=7.6, amide NH), 8.02 (d, IH, J=7.8, amide NH), 7.89 (br s, IH, NH2), 7.71 (d, 2H, J=8.9, aromatic CH), 7.21 (s, IH, aromatic CH), 7.18-7.05 (m, 2H, aromatic CH), 6.98 (d, IH, J=7.4, aromatic CH), 6.79 (d, 2H, J=8.9, aromatic CH), 4.85 (s, 2H, pteridinyl- CH2), 4.39 (m, 2H, methines), 3.23 (s, 3H, N-CH3), 3.03 (m, IH, ArCH2), 2.90 (m, IH, ArCH2), 2.20 (t, 2H, J=7.6, glu 4-CH2), 2.01-1.76 (m, 2H, glu 3-CH2), 1.19 (s, 9H, t-Bu). Elemental Analysis: Calcd. for C33H39N9O6 1.5HC1* 1.8 H2θ (MW 744.85): C, 53.21; H, 5.97; N, 16.92; CI, 7.14. Found: C, 53.20; H, 5.97; N, 16.96; Cl,7.20. Mass Spectrum: (Ion Spray) 658 (M+H) + . UN Spectrum: (pH 7 Buffer) λ max (ε) 259.5(24400), 308.0 (24400), 371.5 (7410). λmin (ε) 241.4 (14600), 274.2 (16200), 345.1 (5680). Example 131: tert-Butyl 5-O-tert-butyl-N-(4-f f ( 1 -2-dihvdro-3-methyl- 1 - oxobenzof f. quinazolin-9-yBmethyπamino V2-fluorobenzoyl'.-L-glutam- 1 -yl-3 - cvclopentyl-L-phenylalaninate A dry 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.150 g of 4-(((l,2-dihydro-3-methyl-l-oxobenzo(f)quinazolin-9- yl)methyl)amino)-2-fluorobenzoic acid, 0.198 g of tert-butyl 5-O-tert-butyl-L-glutam-l- yl-3-cyclopentyl-L-phenylalaninate, and 7 mL of anhydrous DMF. The mixture was cooled to 0°C and treated with 0.058 mL of Et3Ν followed by 0.063 mL of DECP. The solid starting material rapidly dissolved to give a clear solution. The solution was allowed to warm to RT and was stirred at RT under nitrogen. After 3h, tic (Siθ2, 95:5 CH2C_2.MeΟH) indicated a major new component at Rf=0.41 and minor components at Rf=0.38 and 0.06. The solution was concentrated in vacuo to dryness and the residue was dissolved in 70 mL of CHCI3. The CHCI3 solution was washed with 5% aqueous NaHCθ3 (3x60 mL), dried over anhydrous Na2Sθ4 and concentrated to give a yellow glass. The crude product was purified by flash chromatography on silica gel ( 95:5 — > 93:7 CH2Cl2:MeΟH) to afford 0.258 g (80%) of tert-butyl 5-O-tert-butyl-N-(4-(((l,2- dihydro-3-methyl-l-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L- glutam-l-yl-3-cyclopentyl-L-phenylalaninate as a transparent glass. JH-NMR: (300 MHz, DMSO-dό) δ 12.54 (s, IH, benzoquinazoline NH), 9.86 (s, IH, aromatic CH), 8.39 (d, IH, J=7.6, amide NH), 8.23 (d, IH, J=9.0, aromatic CH), 8.02 (d, IH, J=8.4, aromatic CH), 7.68-7.41 (m, 4H, aromatic CH, amide NH), 7.35 (m, IH, 4-aminobenzoyl NH), 7.21-6.97 (m, 4H, aromatic CH), 6.56 (d, IH, J=8.8, aromatic CH), 6.41 (d, IH, J=15.0, aromatic CH), 4.67-4.30 (m, 4H, benzoquinazoline 9-CH2, methines), 3.06-2.77 (m, 3H, ArCH2, cyclopentyl methine), 2.43 (s, 3H, CH3), 2.19 (m, 2H, glu 4-CH2), 2.00-1.38 (m, 10H, glu 3-CH2, cyclopentyl CH2), 1.35 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu). Example 132: N-f4-fffl.2-Dihydro-3-methyl-l-oxobenzoff.quinazolin-9- yπmethvπaminoV 2-fluorobenzoylVL-glutam- 1 -yl-3 -cyclopentyl-L-phenylalanine According to example 50, 0.25 g of tert-butyl 5-O-tert-butyl-N-(4-(((l,2-dihydro-3- methyl-l-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutam-l-yl-3- cyclopentyl-L-phenylalaninate was treated with gaseous HCl in 40 mL of CH3ΝO2 for lh. The reaction mixture was concentrated in vacuo to give a yellow residue. Analysis of this material by HPLC (C18, 65:35:0.1 H2θ:MeCN:TFA) indicated a major component at k'=2.82 and minor components at k'=3.33, 1.53, and 0.83. The crude product was subjected to semi-preparative reverse phase HPLC (C18, 65:35:0.1 H2θ:MeCN:TFA). Fractions containing pure product (as determined by analytical HPLC) were combined and concentrated to dryness. The residue was mixed with 20 mL of water and treated with sufficient IN aqueous NaOH to give complete solution. The solution was filtered and acidified to pH 3.0 by addition of IN aqueous HCl. A white precipitate resulted which was separated by centrifugation, washed with 4 cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 0.141 g (62%) ofN^4-(((l,2-d_hydro-3-methyl-l-oxobe__zo(0quinazolin-9-yl)methyl)amino)-2- fluorobenzoyl)-L-glutam-l-yl-3-cyclopentyl-L-phenylalanine as a fluffy white powder, m.p. >200°C. HPLC: one peak on C18, k'=2.77, 65:55:0.1 H2θ:MeCΝ:TFA, flow rate=l mlVmin. ^-NMR: (300 MHz, DMSO-dό) δ 12.90-11.80 (br m, COOH, benzoquinazoline NH), 9.82 (s, IH, aromatic CH), 8.27 (d, IH, J=7.9, amide NH), 8.19 (d, IH, J=8.7, aromatic CH), 7.99 (d, IH, J=8.4, aromatic CH), 7.58 (m, 2H, aromatic CH), 7.45 (m, 2H, aromatic CH, amide NH), 7.31 (m, IH, 4-aminobenzoyl NH), 7.05 (m, 2H, aromatic CH), 6.97 (d, 2H, J=7.9, aromatic CH), 6.51 (d, IH, J=8.7, aromatic CH), 6.37 (d, IH, J=15.2, aromatic CH), 4.55 (d, 2H, J=5.2, benzoquinazoline 9-CH2), 4.43 (m, 2H, methines), 3.02 (m, IH, ArCH2), 2.82 (m, 2H, ArCH2, cyclopentyl methine), 2.41 (s, 3H, CH3), 2.16 (t, 2H, J=7.7, glu 4-CH2), 1.98-1.71 (m, 4H, glu 3-CH2, cyclopentyl CH 2 ), 1.69-1.31 (m, 6H, cyclopentyl CH2). Elemental Analysis: Calcd. for C40H40N5O7F O.5H2O (MW 730.79): C, 65.74; H, 5.65; N, 9.58. Found: C, 65.76; H, 5.68; N, 9.57. Mass Spectrum: (FAB) 722 (M+H) + . UN Spectrum: (pH 7 Buffer) λmax (ε) 265.9 (47900), 299.6 (27300), 348.4 (5710). λmin (ε) 240.6 (19900), 284.5 (24900), 340.0 (3450). sh (e) 332.3 (6780). Example 133: tert-Butyl N-(4-fff2-amino-3.4-dihydro— 4-oxo-6-quinazolinyπmethvπf2- propynyl')amino'.benzoyl ' .-5-O-tert-butyl-L-glutam-l-yl-3-cvclopentyl-L-phenylalaninate A 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.097 g of 4-(((2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl) (2- propynyl)amino)benzoic acid (Jones, T.R., et al, J. Med. Chem.. 1986, 29, 1114; Νair, M.G., et al, J. Med. Chem.. 1986, 29, 1754; Ghazala, M., et al, J. Med. Chem.. 1986, 29, 1263; Acharya, S.P., et al, J. Heterocvclic Chem.. 1975, 2, 1283), 0.142 g of tert- butyl 5-O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L-phenylalaninate, 0.044 g of 3- hydroxy-l,2,3-benzotriazin-4(3H)-one (Aldrich), and 7 mL of anhydrous DMF. The mixture was treated with 0.057 g of EDC and was stirred at RT under nitrogen. The solid starting material slowly dissolved to give a clear solution. After 3 days the solution was concentrated in vacuo to dryness and the residue was subjected to flash chromatography on silica gel (98:2 → 95:5 CH2C_2:MeOH) to afford 0.142 g (65%) of tert-butyl N-(4-(((2-amino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl)(2- propynyl)amino)benzoyl)-5-O-tert-butyl-L-glutam- 1 -yl-3 -cyclopentyl-L-phenylalaninate as a white solid, m.p. 115°C (dec). Example 134: N**-f4-f((2-Amino-3.4-dihydro-4-oxo-6-quinazolinyDmethyDf2- propynyl ' .amino ' .benzoyl'.-L-glutam-l-yl-3-cvclopentyl-L-phenylalanine According to example 50, 0.114 g of tert-butyl N-(4-(((2-amino-3,4-dihydro-4-oxo-6- quin__zolinyl)methyl)(2-propynyl)amino)benzoyl)-5-O-tert-butyl-L-glut__m- 1 -yl-3 - cyclopentyl-L-phenylalaninate was treated with gaseous HCl in CH3ΝΟ2 for 2h. The reaction mixture was concentrated to dryness and the residue was suspended in 50 mL of ether. The resulting solid was collected by filtration and dried in vacuo. The crude product was purified by semi-preparative reverse phase HPLC (C18, 68:32:0.1 —> 60:40:0.1 H2θ:MeCN:TFA). Fractions containing pure product (as determined by analytical HPLC) were combined and concentrated to dryness. The residue was suspended in water and lyophilized to afford 0.053 g (45%) of N-(4-(((2-amino-3,4- dihydro-4-oxo-6-quinazolinyl)methyl)(2-propynyl)amino)benzoyl)-L-glutam- 1 -yl-3 - cyclopentyl-L-phenylalanine as a fluffy white solid, m.p. 158°C (dec). HPLC: two peaks on C18; k'=1.27 (98.8%), k'=0.50 (1.2%); 62:38:0.1 H2θ:MeCΝ:TFA; flow rate=l mL/min. iH-NMR: (300 MHz, DMSO-dό) δ 12.90-11.80 (br m, COOH), 8.07 (d, IH, J=7.9, amide NH), 8.03 (d, IH, J=7.9, amide NH), 7.87 (s, IH, aromatic CH), 7.71 (d, 2H, J=8.7, aromatic CH), 7.62 (d, IH, J=8.6, aromatic CH), 7.57-7.24 (br s, 2H, NH 2 ), 7.31 (d, IH, J=8.6, aromatic CH), 7.13-6.95 (m, 4H, aromatic CH), 6.82 (d, 2H, J=8.8, aromatic CH), 4.71 (s, 2H, quinazolinyl-CH2), 4.41 (m, 2H, methines), 4.30 (s, 2H, propargyl CH 2 ), 3.19 (s, IH, propargyl CH), 3.10-2.77 (m, 3H, ArCH2, cyclopentyl methine), 2.23 (t, 2H, J=7.6, glu 4-CH 2 ), 2.02-1.76 (m, 4H, glu 3-CH 2 , cyclopentyl CH2), 1.75-1.38 (m, 6H, cyclopentyl CH2). 19 F-NMR: (282 MHz, DMSO-dό) δ 1.53 (relative to TFA external standard). Elemental Analysis: Calcd. for C38H40 6O7 TFA 2.2H2O (MW 846.43): C, 56.76; H, 5.41; N, 9.93. Found: C, 56.79; H, 5.42; N, 9.95. Mass Spectrum: (FAB) 693 (M+H) + . UN Spectrum: (pH 7 Buffer) max (ε) 227.2 (48400), 306.4 (24500). λmin (ε) 213.5 (40200), 252.2 (11500). Example 135: Ethyl N-ffbenzyloxy'.carbonylV5-O-ethyl-L-glutam-l-yl-L- phenylalaninate A 500 mL round bottomed flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 10.0 g of N-benzyloxycarbonyl-L-glutamic acid γ-ethyl ester, 7.43 g of L-phenylalanine ethyl ester hydrochloride (Aldrich), 100 mL of CH2CI2, and 4.51 mL of Et3Ν. The solution was cooled to 0°C and treated with a solution of 6.67 g of DCC (Fluka) in 25 mL of CH2CI2. The solution was stirred at 0°C for lh and then allowed to warm to RT. After an additional 3h, the mixture was filtered to remove dicyclohexylurea. The filtrate was washed with 10% aqueous citric acid (3x50mL), saturated aqueous NaHCO3 (3x50 mL), followed by one 60 mL portion of water. The solution was dried over anhydrous Na2SO4, concentrated to 30 mL, and triturated with addition of 50 mL of ether followed by 150 mL of pentane. The resulting precipitate was collected by filtration and dried in vacuo to afford 12.9 g (82%) of ethyl N- ((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-l-yl-L-phenylalaninate as a white crystalline solid, m.p. 85-87 0 C. Example 136: Ethyl 5-O-ethyl-L-glutam-l-yl-L-phenylalaninate According to example 10, 13.08 g of ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam- 1-yl-L-phenylalaninate was hydrogenolyzed in 300 mL of EtOH using 2.43 g of 10% Pd(C) and 2 mL of acetyl chloride to afford 9.84 g (94%) of ethyl 5-O-ethyl-L-glutam-l- yl-L-phenylalaninate-HCl as a white foam. JH-ΝMR: (200 MHz, DMSO-dό) δ 9.08 (d, IH, J=7.0, ΝH), 7.90-7.40 (br s, 3H, ΝH3 + ), 7.29 (m, 5H, aromatic CH), 4.50 (m, IH, methine), 4.07 (m, 4H, ethyl CH2), 3.80 (m, IH, methine), 3.04 (m, 2H, ArCH2), 2.46 (m, 2H, glu 4-CH2), 1.99 (m, 2H, glu 3-CH2), 1.20 (t, 3H, J=7.1, ethyl CH3), 1.13 (t, 3H, J=7.1, ethyl CH3). Example 137: Ethyl N-(fS -4-fethoxycarbonylV2-f4-nitrophthalimido'.butanoyl'.-L- phenylalaninate A IL round bottomed flask equipped with a magnetic stirrer, a condenser, a nitrogen inlet, and a Dean Stark trap was charged with 7.28 g of ethyl 5-O-ethyl-L-glutam-l-yl-L- phenylalaninate HCl, 3.68 g of 4-nitrophthalic anhydride (American Tokyo Kasei, Portland, OR 97203), 300 mL of toluene, and 2.43 g of dusopropylethylamine (Aldrich). The reaction mixture was heated at reflux for 2h, allowed to cool to RT, and stirred at RT overnight under nitrogen. The toluene was removed by rotary evaporation and the residue was dissolved in 400 mL of CH2CI2. The solution was washed with water (2x200 mL), 5% aqueous ΝaHCθ3 (2x250 mL), and dried over anhydrous MgSO4. The drying agent was removed by filtration and the filtrate was concentrated to 100 mL. The concentrate was mixed with excess ether to induce precipitation. The precipitate was collected by filtration and dried in vacuo to afford 6.72 g (67%) of ethyl N-((S)-4- (ethoxycarbonyl)-2-(4-nitrophthalin_άdo)butanoyl)-L-phenylalaninate as a white solid. 1 H = ΝMR: (300 MHz, DMSO-dό) δ 8.65 (m, 2H, aromatic CH, NH), 8.49 (s, IH, aromatic CH), 8.13 (d, IH, J=8.6, aromatic CH), 7.27-7.06 (m, 5H, aromatic CH), 4.70 (m, IH, methine), 4.39 (m, IH, methine), 4.04 (q, 2H, J=7.1, ethyl CH2), 3.93 (q, 2H, J=7.1, ethyl CH2), 2.94 (dd; IH; J=13.7, 5.8; ArCH2), 2.82 (dd; IH; J=13.6, 9.4; ArCH2), 2.34 (m, 3H, glu 4-CH2, glu 3-CH2), 2.18 (m, IH, glu 3-CH2), 1.11 (t, 6H, J=7.1, ethyl CH3). Example 138: Ethyl N-ffSV2-(4-aminophthalimido')-4-fethoxycarbonv0butanov0-L- phenylalaninate To a 300 mL Parr bottle were added 1.17 g of 10% Pd(C) and 20 mL of EtOAc under a nitrogen flush. To this mixture was added a solution of 6.72 g of ethyl N-((S)-4- (ethoxycarbonyl)-2-(4-nitrophthalimido)butanoyl)-L-phenylalaninate in 200 mL of EtOAc. The mixture was deoxygenated by bubbling nitrogen through and then hydrogenated at 45 psi for lh. The bottle was purged with nitrogen and the catalyst was removed by filtration through celite. The filtrate was concentrated in vacuo to give a residue which was purified by flash chromatography on silica gel (1:1 → 3:7 hexane:EtOAc). This afforded 5.66 g (88%) of ethyl N-((S)-2-(4-aminophthalimido)-4- (ethoxycarbonyl)butanoyl)-L-phenylalaninate as a bright yellow solid. ^-ΝMR: (300 MHz, DMSO-dό) δ 8.50 (d, IH, J=7.4, ΝH), 7.48 (d, IH, J=8.2, aromatic CH), 7.26-7.08 (m, 5H, aromatic CH), 6.92 (s, IH, aromatic CH), 6.82 (dd; IH; J=8.3, 1.6; aromatic CH), 6.47 (s, 2H, ΝH2), 4.54 (m, IH, methine), 4.35 (q, IH, J=7.4, methine), 4.07-3.86 (m, 4H, ethyl CH2), 2.92 (m, 2H, A1-CH2), 2.40-2.13 (m, 4H, glu 4-CH2, glu 3-CH2), 1.09 (m, 6H, ethyl CH3). Example 139: Ethyl N-ffSV2-f 5-amino-l -oxo-2-isoindolinvπ-4- (ethoxycarbonyl ' .butanoylVL-phenylalaninate A Tine-mercury amalgam was prepared by stirring 19.8 g of zinc dust (Mallinckrodt, Inc., Paris, KY, 40361) in 500 mL of 10% aqueous HCl for 10 min. The HCl solution was decanted and the zinc was washed with water until neutral pH was achieved. A warm solution of 0.10 g of HgCl2 in water was added to the zinc and the mixture was stirred for 10 min. The amalgam was collected by filtration, washed consecutively with with water, EtOH, and ether and air dried. The dry amalgam was added to a solution of 3.0 g of ethyl N-((S)-2-(4-aminophthalimido)-4-(ethoxycarbonyl)butanoyl)-L- phenylalaninate and 60 mL of IN ethereal HCl in 250 mL of EtOH which was maintained at 0°C in an ice bath. After stirring at 0°C for 25 min, tic (Siθ2, 3 Al hexane:EtOAc) indicated only a trace of starting material at RfΝ).58 and two new components at Rf=0.52 and 0.63. The reaction mixture was filtered to remove the amalgam and the filtrate was concentrated in vacuo to 200 mL. The solution was hydrogenated at 50 psi in the presence of 0.54 g of 10% Pd(C) for 18h. The catalyst was removed by filtration through celite and the filtrate was neutralized by addition of excess ΝaHC03 and then concentrated to dryness. The residue was partitioned between water and CH2CI2 and the layers were separated. The CH 2 C1 solution was washed with water (2x200 mL) and the water layer was extracted with EtOAc (200 mL). The CH2CI2 and EtOAc solutions were combined, dried over anhydrous Na 2 SO4, and concentrated to give 2.77 g of a pale yellow foam. Analysis of this material by tic (SiO 2 , 9:1 CH 2 Cl 2 :acetone) indicated four major components at Rf-=0.50, 0.41, 0.29, and 0.20. These materials were separated by flash chromatography on silica gel (9: 1 CH 2 C_2:acetone). Comparative ^H-NMR analysis indicated the Rf=0.29 material to be ethyl N-((S)-2-(5-amino- 1 -oxo-2-isoindolinyl)-4-(ethoxycarbonyl)butanoyl)-L- phenylalaninate. The product was obtained as a pale yellow foam, 0.646 g (22%). *H-ΝMR: (300 MHz, DMSO-dό) δ 8.48 (d, IH, J=7.7, ΝH), 7.32 (d, IH, J=8.2, aromatic CH), 7.14 (s, 5H, aromatic CH), 6.59 (m, 2H, aromatic CH), 5.78 (s, 2H, ΝH2), 4.73 (m, IH, methine), 4.43 (m, IH, methine), 4.13-3.90 (m, 6H, ethyl CH2, isoindolinyl methylene), 3.07-2.85 (m, 2H, ArCH2), 2.26-2.01 (m, 3H, glu 4-CH2, glu 3- CH2), 1.88 (m, IH, glu 3-CH2), 1.12 (m, 6H, ethyl CH3). Example 140: Ethyl N-ffSV2-f5-fff 1.2-dihvdro-3-methyl-l-oxobenzoff.quinazolin-9- vOmethvOaminoyi-oxo-2-isoindolinyl -fethoxycarbonyDbutanoylVL-phenylalaninate According to example 5, 1.04 g of 9-bromomethyl-3-methylbenzo(f)quinazolin-l(2H)- one was reacted with 1.65 g of ethyl N-((S)-2-(5-amino-l-oxo-2-isoindolinyl)-4- (ethoxycarbonyl)butanoyl)-L-phenylalaninate using 0.84 g of ΝaHCθ3. The reaction mixture was stirred at 100°C for 5h, cooled to RT, and stirred at RT for 18h. Analysis by tic (Siθ2, 4:1 CH2CI2: acetone) indicated incomplete reaction. An additional 0.3 g of 9-bromomethyl-3-methylbenzo(f)quinazolin-l(2H)-one was added and the reaction mixture was stirred at 100°C for 4h. The mixture was cooled to RT and filtered to remove solids. The crude product was adsorbed onto 25 g of silica gel by adding silica gel to the filtrate, concentrating to dryness, and storing in vacuo. The product/silica gel mixture was loaded onto a column (1000 g Siθ2) and subjected to flash chromatography (CH2CI2 → 95:5 CH 2 Cl2-MeOH) to give 0.30 g of pure product. Impure chromatography fractions were combined, concentrated, and subjected to flash chromatography a second time (98:2 → 95:5 CH 2 Cl :MeOH) to give 0.26 g of pure product . This afforded a total yield of 0.56 g (23%) of ethyl N-((S)-2-(5-(((l,2-dihydro- 3-methyl- 1 -oxobenzo(f)quinazolin-9-yl)methyl)amino)- 1 -oxo-2-isoindolinyl)-4- (ethoxycarbonyl)butanoyl)-L-phenylalaninate as a white solid, m.p. 140°C (dec). Example 141: N-ffS -Carboxy-Σ-fS-fffl.Σ-dihvdro-S-methyl-l- oxobenzofflquinazolin-9-yl'.methyπaminoVI-oxo-2-isoindolinyπbutanoyl'.-L- phenylalanine To a 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 0.90 g of ethyl N-((S)-2-(5-(((l,2-dihydro-3-methyl-l-oxobenzo(f)quinazolin-9- yl)methyl)amino)-l-oxo-2-isoindolinyl)-4-(ethoxycarbonyl)butanoyl)-L-phenylalaninate, 3 mL of EtOH, and 10 mL of 0.2Ν aqueous NaOH. The solution was stirred at RT under nitrogen for 2h and was acidified to pH 3.0 by addition of IN HCl. A white precipitate resulted which was separated by centrifugation. Analysis of this material by analytical HPLC (C18, 70:30:0.10 H2θ:MeCN:TFA) indicated a major component at k-1.93 (90%) and a minor component at k'=2.41. The crude product was purified by semi-preparative reverse phase HPLC (C18, 75:25:0.1 H2θ:MeCN:TFA). Fractions containing pure product (k'=1.93 by analytical HPLC using above conditions) were combined and concentrated in vacuo to dryness. The residue was dissolved in 15 mL of 0. IN aqueous NaOH and the solution was adjusted to pH 3.0 by addition of IN HCl. The resulting precipitate was collected by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, and lyophilized to afford 0.035 g (40%) ofN-((S)-4-C-_rboxy-2-(5-(((l,2-dihydro-3-methyl-l-oxobenzo(f)quinazolin-9- yl)methyl)amino)-l-oxo-2-isoindolinyl)butanoyl)-L-phenylalanine as a white powder, m.p.l80 C. HPLC: one peak on C18, k'=2.3, 70:30:0.10 H2θ:MeCΝ:TFA, flow rate=lmL/min. *H-NMR: (300 MHz, DMSO-dό) δ 12.83-12.56 (br s, IH, COOH), 12.53 (s, IH, benzoquinazoline NH), 12.20-11.90 (br s, IH, COOH), 9.88 (s, IH, aromatic CH), 8.19 (m, 2H, amide NH, aromatic CH), 8.00 (d, IH, J=8.4, aromatic CH), 7.66 (d, IH, J=7.4, aromatic CH), 7.57 (d, IH, J=8.7, aromatic CH), 7.33 (d, IH, J=8.6, aromatic CH), 7.17 (m, IH, isoindolinyl 5-NH), 7.03 (d, 2H, J=7.2, aromatic CH), 6.90 (m, 3H, aromatic CH), 6.72 (d, IH, J=7.8, aromatic CH), 6.59 (s, IH, aromatic CH), 4.67 (m, IH, methine), 4.57 (d, 2H, J=5.9, benzoquinazoline 9-CH2), 4.38 (m, IH, methine), 4.02 (d, IH, J=16.8, isoindolinyl methylene), 3.76 (d, IH, J=16.9, isoindolinyl methylene), 3.00 (m, IH, ArCH2), -80 (m, IH, ArCH2), 2.41 (s, 3H, CH3), 2.02 (m, 3H, glu 4-CH2, glu 3-CH2), 1.76 (m, IH, glu 3-CH2). Elemental Analysis: Calcd. for C36H33N5O7 2.3H2O (MW 689.12): C, 62.75; H, 5.50; N, 10.16. Found: C, 62.82; H, 5.50; N, 10.18. Mass Spectrum: (FAB) 648 (M+H) + . UN Spectrum: (pH 7 Buffer) λ m ax (ε) 265.6 (38900), 301.7 (21100), 348.4 (4870). λmin (ε) 245.1 (19700), 284.1 (18300), 341.8 (3420). sh (e) 331.7 (7570). Example 142: tert-Butyl f2R. 3S. 5S -6-oxo-2.3-diphenyl-5-f3-ftrimethylsilvnbenzvn-4- moφholinecarboxylate A dry 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 2.08 g of t-butyl (2R, 3S)-(-)-6-oxo-2,3-diphenyl-4-moφholinecarboxylate (Aldrich), 1.43 g of 3-(trimethylsilyl)benzyl bromide (Yamakawa, T., et al, J. Med. Chem.. 1990, 33, 1430), and 45 mL of anhydrous THF. The solution was cooled to - 78°C and was treated with 6.17 mL of 1 M sodium bis(trimethylsilyl)amide/THF (Aldrich) via syringe through a rubber septum. The solution was stirred at -78°C for 3.5h and was then poured into 150 mL of water. The resulting mixture was extracted with EtOAc (4x50 mL). The combined EtOAc extracts were washed with saturated brine (3x100 mL), dried over anhydrous Νa2Sθ4, and concentrated in vacuo to give a light yellow oil. The crude material was purified by flash chromatography on silica gel (9:1 hexane:EtOAc) to afford 2.6 g (88%) of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diρhenyl- 5-(3-(trimethylsilyl)benzyl)-4-moφholinecarboxylate as a white powder, m.p. 88-90°C. Example 143: (2R. 3S. 5SV6-Oxo-2-3-diphenyl-5-f3-ftrimethylsilvnbenzyl ' .moφholine According to example 82, 2.47 g of tert-butyl (2R, 3S, 5S 6-oxo-2,3-diρhenyl-5-(3- (trimethylsilyl)benzyl)-4-moφholinecarboxylate was treated with 4.1 mL of TFA in 40 mL of CH2CI2 for 3 h. Neutralization with 8 mL of Et3N and work-up in the usual manner gave a thick oil. Purification of the crude material by flash chromatography on 80 g of silica gel (85:15 hexane.EtOAc) afforded 1.70 g (83%) of (2R, 3S, 5S)-6-oxo- 2,3-diphenyl-5-(3-(trimethylsilyl)benzyl)moφholine as a white crystalline solid, m.p. 94- 95°C. Example 144: tert-Butyl 3-ftrimethylsilylVL-phenylalaninate According to example 53, 1.58 g of (2R, 3S, 5S)-6-oxo-2,3-diρhenyl-5-(3- (trimethylsilyl)benzyl)moφholine was hydrogenolyzed in 70 mL of 1:1 THF:EtOH using 0.70 g of PdC_2- Analysis of the product by *H-NMR indicated partial desilylation of the phenyl ring. The mixture was subjected to esterification according to example 54 using 25 mL of isobutylene and 0.5 mL of cone H2SO4 in 25 mL of 1,4-dioxane. Work-up in the usual manner afforded a light yellow oil which by tic (Siθ2, EtOAc) consisted of two components at Rf=0.53 and 0.47. The Rf-=0.53 material was isolated by flash chromatography twice on silica gel (EtOAc then 1:1 hexane:EtOAc). This gave 0.161 g (15%) of tert-butyl 3-(trimethylsilyl)-L-phenylalaninate as a clear oil. A sample of the corresponding HCl salt was prepared for ^H-NMR by treating 5mg of product with excess IN ethereal HCl and concentrating to dryness. ] H-NMR: (200 MHz, DMSO-dό) δ 8.53 (br s, 3H, NH3 + ), 7.50-7.20 (m, 4H, aromatic CH), 4.14 (m, IH, methine), 3.21 (m, IH, ArCH2), 2.98 (m, IH, ArCH2), 1.26 (s, 9H, t-Bu), 0.23 (s, 9H, trimethylsilyl). Example 145: tert-Butyl N-(fbenzyloxy carbonylV5-O-tert-butyl-L-glutam-l-yl-3- (trimethylsilylVL-phenylalaninate To a 50 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 0.161 g of tert-butyl 3-(trimethylsilyl)-L-phenylalaninate, 0.194 g of N-Cbz-L- glutamic acid γ-tert-butyl ester (Sigma), and 10 mL of CH2CI2. The solution was cooled in to 0°C and was treated with 0.110 g of EDC. The reaction mixture was stirred at 0°C for 1.5h and was then allowed to warm to RT. After an additional lόh of stirring at RT, the solution was concentrated in vacuo to dryness in the presence of 5 g of silica gel. The resulting mixture was applied to a column and the product isolated by flash chromatography (SiΟ 2 , 75:25 hexane:EtΟAc) to afford 0.320 g (95%) of tert-butyl N- ((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-l-yl-3-(trimethylsilyl)-L-phenylalaninate as a thick, transparent glass. -tH-ΝMR: (200 MHz, DMSO-dό) δ 8.29 (d, IH, J=7.0, ΝH), 7.50-7.18 (m, 10H, aromatic CH, ΝH), 5.02 (s, 2H, PhCH 2 O), 4.38 (m, IH, methine), 4.07 (m, IH, methine), 2.97 (d, 2H, J=7.3, ArCH2), 2.23 (t, 2H, J=7.6, glu 4-CH 2 ), 1.96-1.60 (m, 2H, glu 3-CH2), 1.40 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu), 0.24 (s, 9H, trimethylsilyl). Example 146: tert-Butyl 5-O-tert-butyl-L-glut__m-l-yl-3-ftrimethylsilylVL- phenylalaninate According to example 48, 0.315 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl- L-glutam-l-yl-3-(trimethylsilyl)-L-phenylalaninate was hydrogenolyzed using 0.051 g of 10% Pd(C) in 35 mL of MeOH. This afforded 0.24 g (98%) of tert-butyl 5-O-tert-butyl- L-glutam-l-yl-3-(trimethylsilyl)-L-phenylalaninate as a thick transparent oil. A sample of the corresponding HCl salt for ^H-ΝMR analysis was prepared by treating 5 mg of the product with excess IN ethereal HCl and concentrating to dryness. *H-NMR: (200 MHz, DMSO-dό) δ 8.99 (d, IH, J=7.2, NH), 8.28 (br s, 3H, NH3 + ), 7.47-7.24 (m, 4H, aromatic CH), 4.42 (m, IH, methine), 3.86 (m, IH, methine), 3.01 (d, 2H, J=7.2, ArCH2), 2.37 (m, 2H, glu 4-CH2), 2.01 (m, 2H, glu 3-CH2), 1.42 (s, 9H, t- Bu), 1.32 (s, 9H, t-Bu), 0.26 (s, 9H, trimethylsilyl). Example 147: tert-Butyl N-f4-fff2.4-diamino-6- pteridinyπmethyl ' )methylamino'.benzoylV5-O-tert-butyl-L-glutam-l-yl-3-ftrimethylsilylV L-phenylalaninate According to example 49, 0.095 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich) was coupled with 0.12 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3-(trimethylsilyl)-L-phenylalaninate using 0.11 mL of DECP (Aldrich) and 0.12 mL of Et3Ν in 11 mL of DMF. The crude product was subjected to flash chromatography on silica gel (7:7:1 CH2Cl2:acetone:MeΟH) to afford 0.141 g (72%) of tert-butyl N-(4-(((2,4-diamino-6- pteridinyI)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3-(trimethylsilyl)- L-phenylalaninate as a yellow powder, m.p. 118-125°C. JH-ΝMR: (200 MHz, DMSO-dό) δ 8.58 (s, IH, pteridinyl 7-CH), 8.23 (d, IH, J=7.5, amide NH), 8.00 (d, IH, J=7.8, amide NH), 7.74 (d, 2H, J=8.7, aromatic CH), 7.70 (br s, IH, NH2), 7.48 (br s, IH, NH2), 7.33 (m, 2H, aromatic CH), 7.22 (m, 2H, aromatic CH), 6.83 (d, 2H, J=8.8, aromatic CH), 6.63 (br s, 2H, NH2), 4.79 (s, 2H, pteridinyl- CH2), 4.53-4.29 (m, 2H, methines), 3.23 (s, 3H, N-CH3), 2.96 (d, 2H, J=7.0, ArCH2), 2.23 (m, 2H, glu 4-CH2), 2.04-1.77 (m, 2H, glu 3-CH2), 1.38 (s, 9H, t-Bu), 1.28 (s, 9H, t-Bu), 0.21 (s, 9H, trimethylsilyl). Example 148: N-( 4-f f f 2.4-Diamino-6-pteridinvD methyOmethylamino benzoylVL- glutam- 1 -yl-3-f trimethylsilylVL-phenylalanine A 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 0.111 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylaπ_ino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3-(trimethylsilyl)- L-phenylalaninate and 25 mL of CH3ΝO2. The resulting solution was treated with 5 mL of TFA and stirred at RT. After lh, the solution was concentrated in vacuo to dryness. The residue was dissolved in 3 mL of CH3NO2 and the solution was treated with 20 mL of ethyl ether. A yellow solid precipitated which was collected by filtration and dried in vacuo. The product was dissolved in 3 mL of 1 : 1 H2θ:CH3CN and the solution mixed with 50 mL of water. A suspension resulted which was frozen and lyophilized to afford 0.90 g (82%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L- glutam-l-yl-3-(trimethylsilyl)-L-phenylalanine as a yellow powder, m.p. 155°C (dec). HPLC: one peak on C18, k * =1.55, 65:35:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. -1H-NMR: (300 MHz, DMSO-dό) δ 13.00-11.75 (br s, COOH), 8.64 (s, IH, pteridinyl 7-CH), 8.60 (br s, IH, NH2), 8.39 (br s, IH, NH2), 8.11 (d, IH, J=7.7, amide NH), 7.97 (d, IH, J=8.1, amide NH), 7.75-7.10 (br m, 2H, NH2), 7.69 (d, 2H, J=8.9, aromatic CH), 7.28 (m, 2H, aromatic CH), 7.14 (m, 2H, aromatic CH), 6.79 (d, 2H, J=8.9, aromatic CH), 4.82 (s, 2H, pteridinyl-CH2), 4.39 (m, 2H, methines), 3.21 (s, 3H, N- CH3), 3.02 (m, IH, ArCH2), 2.88 (m, IH, ArCH2), 2.20 (t, 2H, J=7.8, glu 4-CH2), 2.00-1.72 (m, 2H, glu 3-CH2), 0.17 (s, 9H, trimethylsilyl). Elemental Analysis: Calcd. for C32H39 9θόSi-0.8 TFA 1.2 H2O (MW 786.64): C, 51.30; H, 5.41; N, 16.03. Found: C, 51.24; H, 5.39; N, 16.07. Mass Spectrum: (FAB) 674 (M+H) + UN Spectrum: (pH 7 Buffer) λ m ax (ε) 259.5 (23500), 308.0 (24300), 373.2 (7210). λmin (ε) 240.7 (12700), 273.9 (15500), 345.0 (5210). Example 149: tert-Butyl 3-iodo-L-tyrosinate A mixture of 5.0 g of 3-iodo-L-tyrosine (Aldrich) and 2.5 mL of cone H2SO4 in 85 mL of 1,4-dioxane was added to 30 mL of liquid isobutylene (condensed at -78°C) in a 300 mL pressure bottle. The vessel was stoppered and the contents stirred at RT. After 3 days the bottle was cooled in an ice water bath, opened and the solution poured into a mixture of 10 g of NaHCO3 in 150 mL of water. The resulting mixture was subjected to rotary evaporation to remove isobutylene and dioxane. A white solid resulted which was collected by filtration and dried in vacuo. Analysis by tic (Siθ2, 95:5 CH2Cl2-MeOH) indicated major and minor components at Rf =0.52 and 0.60 respectively. The crude product was purified by flash chromatography on silica gel (95:5 CH2θ2:MeOH) to afford 3.4 g (58%) of tert-butyl 3-iodo-L-tyrosinate as a white crystalline solid, m.p. 158-159 0 C. Example 150: tert-Butyl N-(fbenzyloxy carbonyl>-4-O-ffbenzyloxy'.carbonylV3-iodo-L- tyrosinate A 500 mL round bottomed flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 5.15 g of tert-butyl 3-iodo-L-tyrosinate, 250 mL of 1,4-dioxane, and 4.55 mL of Et3Ν. The resulting solution was treated with 4.66 mL of benzyl chloroformate by slow addition. After 30 min, tic (Siθ2, 8:2 hexane:EtOAc) indicated no remaining starting material and a single new component at Rf=0.50. The solution was concentrated in vacuo and the residue mixed with 200 mL of water. The mixture was extracted with EtOAc (4x50mL). The combined EtOAc extracts were washed with 5% aqueous NaHCO3, dried over anhydrous MgSO4, and concentrated in vacuo. The crude product was subjected to flash chromatography on silica gel (8:2 hexane:EtOAc) to afford 7.49 g (84%) of tert-butyl N* (benzyloxy)carbonyI)-4-O-((benzyloxy)carbonyl)-3- iodo-L-tyrosinate as a transparent foam. JH-ΝMR: (200 MHz, DMSO-d6) δ 7.77 (m, 2H, ΝH, aromatic CH), 7.53-7.21 (m, 12H, aromatic CH), 5.32 (s, 2H, PhCH2θ), 5.20 (s, 2H, PhCH2θ), 4.12 (m, IH, methine), 2.92 (m, 2H, ArCH2), l- 5 (s, 9H, t-Bu). Example 151: tert-Butyl 3 -cyclopentyl-L-tyrosinate A 300 mL Parr bomb was charged with 5.13 g of tert-butyl Ν-((benzyloxy)carbonyl)-4- O-((benzyloxy)carbonyl)-3-iodo-L-tyrosinate, 120 mL of toluene, 30 mL of cyclopentene and 0.49 g of tri-ortho-tolylphosphine. The solution was deoxygenated by bubbling nitrogen through for 10 min and was treated with 0.182 g of Pd(OAc) 2 and 1.24 mL of Et3N. The bomb was flushed with nitrogen, sealed, and heated to 110°C for 48h. The vessel was cooled to RT, purged with nitrogen and opened. The reaction mixture was filtered to remove solids and the filtrate was concentrated in vacuo to dryness. The residue was dissolved in 200 mL of EtOAc and the solution was washed with water (4x100 mL), dried over anhydrous NaSO4 and concentrated to give a thick brown oil. Analysis by tic (SiO 2 , 8:2 hexane:EtOAc) indicated two major componenets at Rf=0.50 and 0.40. The crude mixture was subjected to flash chromatography on silica gel (8:2 hexane :EtO Ac). This afforded 2.0 g (43%) of the Rf=0.50 material identified as the bis(benzyloxycarbonyl) olefin mixture by -1H-NMR. In addition, 0.90 g (25%) of the Rf=0.40 material was obtained and was identified as the N-benzyloxycarbonyl olefin mixture resulting from loss of the O-benzyloxycarbonyl group. The bis(benzyloxycarbonyl) olefin mixture (2.00 g) was subjected to catalytic hydrogenation (45 psi) in the presence of 0.35 g of 10% Pd(C) in 70 mL of MeOH for 18h. The crude product was purified by flash chromatography (Siθ2, EtOAc) to give 0.99 g (93%) of tert-butyl 3-cyclopentyl-L-tyrosinate as a transparent glass. The N-benzyloxycarbonyl olefin mixture (0.90 g) was similarly hydrogenated and purified to afford 0.52 g (83%) of tert-butyl 3-cyclopentyl-L-tyrosinate. -NMR: (200 MHz, DMSO-dό) δ 9.05 (s, IH, OH), 6.92 (d, IH, J=1.9, aromatic CH), 6.80 (dd; IH; J=8.0, 2.0; aromatic CH), 6.66 (d, IH, J=8.0, aromatic CH), 3.16 (m, IH, cyclopentyl methine), 2.67 (d, 2H, J=6.6, ArCH2), 2.00-1.40 (m, 10H, cyclopentyl CH2, NH2), 1.33 (s, 9H, t-Bu). Example 152: tert-Butyl N-f(benzyloxy carbonyl)-5-O-tert-butyl-L-glutam-l-yl-3- cvclopentyl-L-tyrosinate According to example 145, 0.99 g of tert-butyl 3-cyclopentyl-L-tyτosinate was coupled with 1.09 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.65 g of EDC in 20 mL of CH2CI2 for 4h. The crude product was purified by flash chromatography on silica gel (7:3 hexane:EtOAc) to afford 1.8 g (89%) of tert-butyl N- ((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam- 1 -yl-3-cyclopentyl-L-tyrosinate as a colorless foam. !H-ΝMR: (200 MHz, DMSO-dό) δ 9.08 (s, IH, OH), 8.17 (d, IH, J-=7.5, NH), 7.36 (m, 6H, aromatic CH, NH), 6.96 (s, IH, aromatic CH), 6.81 (d, IH, J=8.1, aromatic CH), 6.67 (d, IH, J=8.1, aromatic CH), 5.01 (m, 2H, PhCH2θ), 4.28 (m, IH, methine), 4.08 (m, IH, methine), 3.17 (m, IH, cyclopentyl methine), 2.82 (d, 2H, J=6.9, ArCH2), 2.21 (t, 2H, J=7.7, glu 4-CH2), 1.99-1.46 (m, 10H, glu 3-CH2, cyclopentyl CH2), 1.40 (s, 9H, t-Bu), 1.33 (s, 9H, t-Bu). Example 153: tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-3-cvclopentyl-L-tyrosinate According to example 48, 1.75 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L- glutam-l-yl-3-cyclopentyl-L-tyrosinate was hydrogenolyzed using 0.28 g of 10% Pd(C) in 70 mL of MeOH for 3h. The crude product was subjected to flash chromatography on silica gel (95:5 CH2Cl2:MeOH) to afford 1.26 g (92%) of tert-butyl 5-O-tert-butyl- L-glutam-l-yl-3-cyclopentyl-L-tyτosinate as a white crystalline solid, m.p. 108-110°C. Example 154: tert-Butyl N-f4-fff 2.4-diamino-6- pteridinvnmethvnmethylamino^benzovn-S-O-tert-butyl-L-glutam-l-yl-S-cvclopentyl-L- tyrosinate To a dry 100 mL 3 -necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 35 mL of anhydrous DMF, 1.14 mL of Et3Ν, and 0.99 mL of DECP. To the resulting solution were added 1.24 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate (Aldrich). The solid starting material slowly dissolved to give a yellow solution. After 45 min, a solution of 1.60 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L-tyrosinate in 8 mL of DMF was added. The solution was stirred at RT for 24h and was then concentrated in vacuo to dryness. The residue was dissolved in 150 mL of CHCI3. The solution was washed with 5% aqueous ΝH4ΟH (3x70 mL), dried over anhydrous MgSO4, and concentrated in vacuo. The crude product was subjected to flash chromatography on silica gel twice (7:7:1 CH2Cl 2 :acetone:MeOH, 93:7 CH2C_2:MeOH) to afford 2.08 g (80%) of tert- butyl N*^4-(((2,4-di__mino-6-pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L- glutam-l-yl-3-cyclopentyl-L-tyrosinate as a yellow powder, m.p. 145-148°C. Example 155: N*^4-(((2.4-Dianτino-6-pteridinyl)methyl)methylamino benzoyQ-L- glutam-1 -yl-3-cyclopentyl-L-tyrosine According to example 50, 2.04 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3-cyclopentyl-L- tyrosinate was treated with gaseous HCl in 65 mL of CH3ΝO2 for lh. The yellow suspension was concentrated to a slurry and mixed with 80 mL of ethyl ether. The resulting precipitate was collected by filtration and dried in vacuo. The product was dissolved in 40 mL of 8:2 H2θ:MeCN and the solution was filtered and concentrated to 30 mL. A yellow suspension was obtained which was diluted with 150 mL of water, frozen, and lyophilized to give 1.71 g (86%) ofN-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3-cyclopentyl-L-tyrosine as a yellow-orange powder, m.p. 175°C (dec). HPLC: one peak on C18, k'=2.98, 75:25:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. H-NMR: (300 MHz, DMSO-d6) δ 13.12 (br m, COOH), 9.30 (s, IH, NH2), 9.09 (s, IH, NH2), 8.71 (s, IH, pteridinyl 7-CH), 8.67 (br s, IH, NH2), 8.02 (m, 2H, amide NHs), 7.78 (br s, IH, NH 2 ), 7.71 (d, 2H, J=8.7, aromatic CH), 6.91 (s, IH, aromatic CH), 6.79 (m, 3H, aromatic CH), 6.61 (d, IH, J=8.1, aromatic CH), 4.86 (s, 2H, pteridinyl-CH2), 4.42 (m, IH, methine), 4.29 (m, IH, methine), 3.24 (s, 3H, N-CH3), 3.09 (m, IH, cyclopentyl methine), 2.93-2.71 (m, 2H, ArCH2), 2.23 (t, 2H, J=7.8, glu 4- CH2), 2.03-1.74 (m, 4H, glu 3-CH2, cyclopentyl CH2), 1.73-1.37 (m, 6H, cyclopentyl CH 2 ). Elemental Analysis: Calcd. for C34H39N9O7- 1.6 HCl* 1.7 H 2 O (MW 774.70): C, 52.71; H, 5.72; N, 16.27; CI, 7.32. Found: C, 52.79; H, 5.71; N, 16.16; CI, 7.35. Mass Spectrum: (Ion Spray) 686 (M+H) + . UN Spectrum: (pH 7 Buffer) λ m ax (ε) 259.3 (24700), 307.8 (24800), 372.5 (7780). λmin (ε) 24 2.3 (15500), 272.9 (17800), 344.6 (6080). sh (e) 221.3 (28900), 286.4 (21100). Example 156: 3-f3-hvdroxy-3-pentylVO-ftert-butyltrimethylsilyl')benzyl alcohol To a dry 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 5.0 g of 3-iodo-O-(tert-butyldimethylsilyl)benzyl alcohol and 40 mL of anhydrous ethyl ether. The solution was cooled to -78°C under nitrogen and was treated with 23.2 mL of 1.3 M sec-butyllithium/cyclohexane (Aldrich) via syringe. After 20 min, the solution was treated with 3.1 mL of 3-pentanone (Aldrich). The reaction mixture was stirred at -78°C for 1.5h and was then allowed to warm to RT. After 3h at RT the solution was quenched by addition of 5 mL of water, stirred for several minutes and then mixed with 80 mL of water. The two phases were separated and the aqueous solution was extracted with an additional 50 mL portion of ether. The ether extract was combined with the original ether solution. This solution was washed with saturated aqueous brine (3x50 mL), dried over anhydrous MgSO4, and concentrated to afford a clear liquid. Analysis by tic (SiO 2 , 9: 1 hexane:EtOAc) indicated a major new component at R =0.47. The crude product was purified by flash chromatography on silica gel (9: 1 hexane:EtOAc) to afford 3.9 g (88%) of 3-(3-hydroxy-3-pentyl)-O-(tert- butyltrimethylsilyl)benzyl alcohol as a clear liquid. *H-NMR: (200 MHz, DMSO-dό) δ 7.33 (s, IH, aromatic CH), 7.26 (m, 2H, aromatic CH), 7.12 (m, IH, aromatic CH), 4.72 (s, 2H, ArCH 2 O), 4.48 (s, IH, OH), 1.72 (m, 4H, pentyl CH 2 ), 0.90 (s, 9H, t-Bu), 0.64 (t, 6H, J=7.5, pentyl CH3), 0.06 (s, 6H, SiMe 2 ). Example 157: 3-f3-Pentyl)benzyl alcohol A 250 mL round bottomed flask equipped with a nitrogen inlet and a magnetic stirrer was charged with 3.9 g of 3-(3-hydroxy-3-pentyl)-O-(tert-butyltrimethylsilyl)benzyl alcohol and 25 mL of CH 2 Cl2- The solution was cooled to 0°C in an ice bath and was treated with 15 mL of TFA followed by 5 mL of Et3SiH (Aldrich). After 30 min at 0°C, the ice bath was removed and the solution was stirred at RT for 18h. The solution was concentrated to a viscous liquid. This material was dissolved in ether (60 mL), washed with 5% aqueous NaHCθ3 (3x50 mL), dried over anhydrous MgSθ4, and concentrated in vacuo. The resulting liquid was dissolved in 10 mL of THF and treated with 63 mL of IM BU4NF/THF (Aldrich). After stirring at RT for 18h, the solution was concentrated and the resulting syrup mixed with 80 mL of water. The mixture was extracted with ether (4x30 mL). The combined extracts were washed with 5% aqueous NaHCO3 (3x50 mL), dried over anhydrous MgSO4 > and concentrated to afford a light yellow liquid. Analysis by tic (Siθ2, 9:1 hexane:EtOAc) indicated a major new component at Rf=0.30. The crude product was purified by flash chromatography on silica gel (85:15 hexane:EtOAc) to give 1.84 g (82%) of 3-(3-pentyl)benzyl alcohol as a clear liquid. JH-NMR: (300 MHz, DMSO-dό) δ 7.21 (t, IH, J=7.5, aromatic CH), 7.10 (m, 2H, aromatic CH), 6.99 (d, IH, J=7.4, aromatic CH), 5.12 (t, IH, J=5.8, OH), 4.46 (d, 2H, J=5.8, ArCH 2 O), 2.26 (m, IH, pentyl methine), 1.62 (m, 2H, pentyl CH ), 1.49 (m, 2H, pentyl CH 2 ), 0.69 (t, 6H, J=7.4, pentyl CH3). Example 158: 3-f3-Pentvπbenzyl bromide According to example 17, 1.84 g of 3-(3-pentyl)benzyl alcohol was treated with 0.34 mL of PB13 in 25 mL of anhydrous CH 2 C1 2 to afford 2.23 g (90%) of 3-(3-Pentyl)benzyl bromide as a clear liquid. iH-NMR. (200 MHz, DMSO-d6) δ 7.26 (m, 3H, aromatic CH), 7.12 (m, IH, aromatic CH), 4.70 (s, 2H, ArCH2), 2.33 (m, IH, pentyl methine), 1.78-1.40 (m, 4H, pentyl CH2), 0.73 (t, 6H, J=7.3, pentyl CH3). Example 159: tert-Butyl f2R. 3S. 5S.-6-oxo-2.3-diphenyl-5-f3-f3-pentyl'.benzvn-4- moφholinecarboxylate According to example 142, 3.27 g of tert-butyl (2R, 3S (-)-6-oxo-2,3-diphenyl-4- moφholinecarboxylate was alkylated with 2.23 g of 3-(3-pentyl)benzyl bromide in 70 mL of anhydrous THF using 9.71 mL of 1 M sodium bis(trimethylsilyl)amide in THF. The crude product was subjected to flash chromatography on silica gel (9:1 hexane:EtOAc) and then recrystallization from EtOH-water to afford 2.26 g (48%) of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(3-pentyl)benzyl)-4- moφholinecarboxylate as a white crystalline solid, m.p. 143-144°C. Example 160: (2R. 3S. 5S -6-Oxo-2.3-diphenyl-5-f3-f3-pentyl)benzvnmoφholine To a 250 mL round bottomed flask equipped with a magnetic stirrer and a nitrogen inlet were added 2.19 g of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-(3-pentyl)benzyl)- 4-moφholinecarboxylate and 45 mL of anhydrous CH2CI2. The solution was cooled to 0°C and was treated with 3.5 mL of TFA. The solution was allowed to warm to RT and was stirred under nitrogen. After 5h, tic (Siθ2, 9:1 hexane:EtOAc) indicated a trace of starting material and a major new component at Rf=0.42. The reaction mixture was poured into 150 mL of 6% aqueous NaHCO3 and stirred for several minutes. The phases were separated and the aqueous portion was extracted with 3 additional 40 mL portions of CH2CI2. The extracts were combined with the original CH2CI2 solution and washed with 5% aqueous NaHCO3 (3x60mL). The solution was dried over anhydrous MgSO4 and concentrated to give a light tan solid. This material was purified by flash chromatography on silica gel (85:15 hexane:EtOAc) to afford 1.47 g (84%) of (2R, 3S, 5-_ -6-oxo-2,3-diphenyl-5-(3-(3-pentyl)benzyl)moφholine as a white crystalline solid. An analytical sample was prepared by recrystallization from EtOH-H2θ. Melting point, 96-97°C. Example 161: 3 -f 3 -Pentyl) -L-phenylalanine To a 300 mL Parr bottle were added 0.41 g of PdCl 2 , 1.36 g of (2R, 3S, 5S)-6-oxo-2,3- diphenyl-5-(3-(3-pentyl)benzyl)moφholine, 50 mL of EtOH, and 30 mL of THF. The mixture was deoxygenated by bubbling nitrogen through for lOmin and was hydrogenated at 45 psi for 18h. The vessel was purged with nitrogen, catalyst removed by filtration through celite, and the filtrate concentrated in vacuo to a volume of 15 mL. The solution was chilled in an ice bath and triturated with addition of 50 mL of hexane. A fine precipitate slowly formed. After storing at 5°C overnight, the solid was collected by filtration and dried in vacuo to afford 0.65 g (72%) of 3-(3-pentyl)-L- phenylalanine HCl as a white powder, m.p. 170°C (dec). Example 162: tert-Butyl 3-f3-pentylVL-phenylalaninate According to example 54, 0.60 g of 3-(3-pentyl)-L-phenylalanine-HCl was treated with 30 mL of isobutylene in 35 mL of 1,4-dioxane in the presence of 0.5 mL of cone H 2 SO4. Work-up in the usual manner followed by acidification with 2 mL of IN ethereal HCl afforded 0.53 g (74%) of tert-butyl 3-(3-pentyl)-L-phenylalaninate-HCl as a light yellow glass. *H-NMR: (200 MHz, DMSO-dό) δ 8.63 (br s, 3H, NH3 + ), 7.28 (t, IH, J=7.45, aromatic CH), 7.10 (m, 3H, aromatic CH), 4.12 (m, IH, methine), 3.23 (dd; IH; J=13.9, 5.1; ArCH2), 2.96 (dd; IH; J=13.9, 9.1; ArCH2), 2.32 (m, IH, pentyl methine), 1.78- 1.40 (m, 4H, pentyl CH2), 1-28 (s, 9H, t-Bu), 0.74 (t, 6H, J=7.3, pentyl CH3). Example 163: tert-Butyl N- benzyloxy. carbonyl V 5-O-tert-butyl-L-glutam- 1 -yl-3 -f 3 - pentylVL-phenylalaninate According to example 145, 0.53 g of tert-butyl 3-(3-pentyl)-L-phenylalaninate*HCl was coupled with 0.55 g of N-Cbz-L-glutamic acid γ-tert-butyl ester using 0.33 g of EDC and 0.18 mL ofN-methylmoφholine in 15 mL of anhydrous CH2CI2. The crude product was purified by flash chromatography on silica gel (8:2 hexane: EtOAc) to afford 0.80 g (81%) of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-l-yl-3-(3-pentyl)- L-phenylalaninate as a white crystalline solid, m.p. 50-53°C. Example 164: tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-3-f3-pentvπ-L-phenylalaninate According to example 48, 0.72 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L- glutam-l-yl-3-(3-pentyl)-L-phenylalaninate was hydrogenolyzed using 0.12 g of 10% Pd(C) in 70 mL of MeOH. This afforded 0.51 g (91%) of tert-butyl 5-O-tert-butyl-L- glutam-l-yl-3-(3-pentyl)-L-phenylalaninate as a thick transparent oil. A sample of the corresponding HCl salt for *1H-ΝMR analysis was prepared by treating 5 mg of the product with excess IN ethereal HCl and concentrating to dryness. Ϊ H-NMR: (200 MHz, DMSO-dό) δ 9.03 (d, IH, J=6.9, NH), 8.38 (br s, 3H, NH3 + ), 7.23 (t, IH, J=7.2, aromatic CH), 7.06 (m, 3H, aromatic CH), 4.39 (m, IH, methine), 3.87 (m, IH, methine), 3.01 (d, 2H, J=7.4, ArCH2), 2.35 (m, 3H, pentyl methine, glu 4- CH 2 ), 2.01 (m, 2H, glu 3-CH2), 1.75-1.48 (m, 4H, pentyl CH2), 1.41 (s, 9H, t-Bu), 1.32 (s, 9H, t-Bu), 0.73 (t, 6H, J=7.3, pentyl CH3). Example 165: tert-Butyl N-t/4-fff 2.4-diamino-6- pteridinyl)methyπmethylamino)benzoyπ-5-O-tert-butyl-L-glutam-l-yl-3-f3-pentyπ-L- phenylalaninate According to example 49, 0.199 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate was coupled with 0.25 g of tert- butyl 5-O-tert-butyl-L-glutam-l-yl-3-(3-pentyl)-L-phenylalaninate using 0.24 mL of DECP and 0.26 mL of Et3Ν in 25 mL of DMF. The crude product was subjected to flash chromatography twice on silica gel (7:7:1 CH2θ2:acetone:MeΟH; 95:5 CH2Cl2:MeΟH ) to afford 0.215 g (52%) of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3-(3-pentyl)-L- phenylalaninate as a yellow powder, m.p. 175°C (dec). Example 166: N- 4-f f f2.4-Diamino-6-pteridinv0methyl)methylamino)benzoylVL- glutam-l-yl-3-f3-pentylVL-phenylalanine According to example 50, 0.184 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3-(3-pentyl)-L- phenylalaninate was treated with gaseous HCl in 25 mL of CH3ΝO2 for 45 min. The yellow suspension was concentrated in vacuo to dryness. Analysis of the product by HPLC (C18, 65:35:0.1 H2θ:MeCN:TFA) indicated a major component at k'=1.61 and a minor component at k'=0.25. The crude product was purified by semi-preparative HPLC (C18, 65:35:0.1 H2θ:MeCN:TFA). Fractions containing pure product (analytical HPLC) were combined and subjected to rotary evaporation to remove CH3CN. A yellow suspension resulted which was frozen and lyophilized to afford 0.12 g (58%) of *^ 4 -(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3-(3- pentyl)-L-phenylalanine as a yellow powder, m.p. 120°C (dec). HPLC: one peak on C18, k'=1.61, 65:35:0.1 H2θ:MeCN:TFA, flow rate=l mlJmin. Ϊ H-NMR: (300 MHz, DMSO-dό) δ 12.87-11.90 (br m, 2H, COOH), 9.20 (br s, IH, NH2), 9.00 (br s, IH, NH2), 8.70 (s, IH, pteridinyl 7-CH), 8.60-8.23 (br m, 2H, NH2), 8.11 (d, IH, J=7.6, amide NH), 7.96 (d, IH, J=7.8, amide NH), 7.70 (d, 2H, J=8.7, aromatic CH), 7.07 (m, IH, aromatic CH), 6.98 (d, 2H, J=7.9, aromatic CH), 6.90 (d, IH, J=7.6, aromatic CH), 6.80 (d, 2H, J=8.6, aromatic CH), 4.86 (s, 2H, pteridinyl- CH2), 4.41 (m, 2H, methines), 3.23 (s, 3H, N-CH3), 2.92 (m, 2H, ArCH2), 2.21 (m, 3H, pentyl methine, glu 4-CH2), 2.03-1.77 (m, 2H, glu 3-CH2), 1.68-1.37 (m, 4H, pentyl CH2), 0.62 (m, 6H, pentyl CH3). Elemental Analysis: Calcd. for C34H41N9O6T.5TF A- 1.8 H2O (MW 875.22): C, 50.78; H, 5.31; N, 14.40. Found: C, 50.76; H, 5.40; N, 14.34. Mass Spectrum: (Ion Spray) 672 (M+H) + . UN Spectrum: (pH 7 Buffer) λmax (ε) 259.8 (20000), 308.7 (19800), 373.7 (5890). λmin (ε) 241.5 (11600), 275.0 (13200), 345.8 (4420). Example 167: 2-Iodo-4-nitro-L-phenylalanine A 250 mL round bottomed flask equipped with a magnetic stirrer was charged with 60 mL of fuming sulfuric acid (Aldrich) and 12.1 g of iodine (Aldrich). To the resulting dark red solution was added 10.0 g of 4-nitro-L-phenylalanine by slow addition. The flask was fitted with a CaCl2 drying tube and the reaction mixture was stirred at RT for 18h. The mixture was then poured onto 300 g of crushed ice. A yellow-brown emulsion resulted which was treated with 7.4 g of sodium thiosulfate and stirred. The resulting light brown suspension was basified to pH=9 by addition of solid NaCO3. This was followed by acidification to pH=5.5 by addition of cone H2SO4. A brown solid precipitated which was collected by filtration and dried in vacuo. This afforded 6.54 g (41%) of 2-iodo-4-nitro-L-phenylalanine as a brown powder, m.p.>200°C. *H-NMR: (200 MHz, DMSO-dό/TFA) d 8.62 (s, IH, aromatic CH), 8.46 (br s, 3H, NH3+), 8.26 (d, IH, J=8.5, aromatic CH), 7.65 (d, IH, J=8.5, aromatic CH), 4.22 (m, IH, methine), 3.34 (m, 2H, ArCH2). Example 168: Ethyl 2-iodo-4-nitro-L-phenylalaninate According to example 8, 1.40 g of 2-iodo-4-nitro-L-phenylalanine was esterified by refluxing in 50 mL of ethanolic HCl for 18h. Work-up in the usual manner followed by flash chromatography on silica gel (95:5 CH2Cl2.MeOH) afforded 1.19 g (78%) of ethyl 2-iodo-4-nitro-L-phenylalaninate as a yellow crystalline solid, m.p.33-34°C. Example 169: Ethyl N-ffbenzyloxy)carbonylV2-iodo-4-nitro-L-phenylalaninate To a 250 mL round bottomed flask equipped with a magnetic stirrer and a nitrogen inlet were added 4.7 g of ethyl 2-iodo-4-nitro-L-phenylalaninate, 125 mL of 1,4-dioxane, and 2.2 mL of Et3N. The solution was cooled in an ice water bath and treated with 2.2 mL of benzyl chloroformate by slow addition. The ice bath was removed and the reaction mixture was stirred at RT for 18h. The mixture was filtered and the filtrate was concentrated to dryness. The brown residue was dissolved in EtOAc, washed with 5% aqueous NaHCO3 (4x75 mL), dried over anhydrous MgSO4 and concentrated in vacuo. Analysis of the crude product by tic (Siθ2, 75:25 hexane:EtOAc) indicated a major new component at Rf=0 * 35 and minor components at Rf-Φ.40 and 0.25. The R =0.35 product was isolated by flash chromatography on silica gel (75:25 hexane :EtO Ac). This afforded 2.08 g (32%) of ethyl N-((benzyloxy)carbonyl)-2-iodo-4-nitro-L- phenylalaninate as a white powder, m.p. 85-87°C. Example 170: Ethyl 4-amino-2-cyclopentyl-L-phenylalaninate According to example 151, 1.65 g of ethyl N-((benzyloxy)carbonyl)-2-iodo-4-nitro-L- phenylalaninate was subjected to Heck coupling with 20 mL of cyclopentene using 60 mL of toluene, 0.074 g of Pd(OAc)2, 0.20 g of tri-ortho-tolylphosphine, and 0.51 mL of Et3Ν. The reaction was carried out at 110°C for 24h. Analysis of the crude product by tic (Siθ2, 8:2 hexane:EtOAc) indicated a major product at Rf=0.40 and minor components at Rf=0.38, 0.60, and 0.95. The Rf=0.40 material was isolated by flash chromatography on silica gel (8:2 hexane:EtOAc). This afforded 1.03 g (71%) of the desired olefin mixture as a clear liquid. This material was subjected to catalytic hydrogenation at 45 psi for 18h using 55 mL of MeOH and 0.25 g of 10% Pd(C). The crude product was purified by flash chromatography (Siθ2, 99:1 EtOAcMeOH) to give 0.56 g (61% for both steps) of ethyl 4-amino-2-cyclopentyl-L-phenylalaninate as a thick transparent oil. ^-NMR: (200 MHz, DMSO-dό) δ 6.72 (d, IH, J=8.2, aromatic CH), 6.50 (d, IH, J=2.3, aromatic CH), 6.29 (dd; IH; J=8.1, 2.3; aromatic CH), 4.80 (br s, 2H, aromatic NH2), 4.00 (q, 2H, J=7.1, ethyl CH2), 3.12 (m, IH, cyclopentyl methine), 2.72 (m, 2H, ArCH2), 2.03-1.37 (m, 10H, cyclopentyl CH2, NH2), 1.11 (t, 3H, J=7.1, ethyl CH3). Example 171: Ethyl 2-cvclopentyl-L-tyrosinate To a 500 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer were added a solution of 0.56 g of ethyl 4-amino-2-cyclopentyl-L-phenylalaninate in 60 mL of 0.1 M aqueous H2SO4. After cooling to 3°C, the solution was treated with a solution of 0.148 g of sodium nitrite (Aldrich) in 10 mL of water. The solution was stirred at 3°C for 10 min and was then treated with 95 mL of 1.5 M aqueous Q1SO4 followed by 0.25 g of Cu2θ (Aldrich). The mixture was heated to 45°C for 35 min with stirring. Gas evolution was evident during this period. The mixture was cooled to RT and filtered. The blue filtrate was treated with IN aqueous NaOH to pH=5.5. The resulting blue- green suspension was transfered to a separatory funnel and extracted with CHCI3 (5x100 mL). The combined CHCI3 extracts were dried over MgSO4 and concentrated to give 0.12 g of a green oil. The blue-green suspension was then treated with solid NaHCO3 until gas evolution ceased and was extracted with CHCI3 a second time (4x50 mL). The combined extracts were washed with water, dried (MgSO4), and concentrated to give 0.076 g of an oil. This material was combined with the original 0.12 g of product and purified by flash chromatography (Siθ2, 95:5 CH2Cl2:MeOH) to afford 0.168 g (30%) of ethyl 2-cyclopentyl-L-tyrosinate as a thick, transparent oil. H-NMR: (200 MHz, DMSO-dό) δ 9.07 (s, IH, OH), 6.87 (d, IH, J=8.2, aromatic CH), 6.66 (d, IH, J=2.4, aromatic CH), 6.48 (dd; IH; J=8.1, 2.4; aromatic CH), 4.00 (q, 2H, J=7.1, ethyl CH2), 3.16 (m, IH, cyclopentyl methine), 2.78 (m, 2H, ArCH2), 2.05- 1.32 (m, 10H, cyclopentyl CH2, NH2), 1.10 (t, 3H, J=7.1, ethyl CH3). Example 172: Ethyl N-ffbenzyloxy)carbonyl'>-5-O-ethyl-L-glutam-l-yl-2-cyclopentyl-L- tyrosinate According to example 145, 0.153 g of ethyl 2-cyclopentyl-L-tyrosinate was coupled with 0.171 g of N-Cbz-L-glutamic acid γ-ethyl ester using 0.111 g of EDC in 8 mL of CH2CI2 for 4.5h. The crude product was purified by flash chromatography twice on silica gel (1:1 hexane:EtOAc; 95:5 CH2C_2:MeOH) to afford 0.245 g (78%) of ethy! N- ((benzyloxy)carbonyl)-5-O-ethyl-L-glutam-l-yl-2-cyclopentyl-L-tyrosinate as a colorless foam. H-ΝMR: (300 MHz, DMSO-dό) δ 9.09 (s, IH, OH), 8.37 (d, IH, J=7.3 ΝH), 7.33 (m, 6H, aromatic CH, ΝH), 6.88 (d, IH, J=8.4, aromatic CH), 6.63 (s, IH, aromatic CH), 6.43 (d, IH, J=8.3, aromatic CH), 4.99 (m, 2H, PhCH2θ), 4.28 (m, IH, methine), 4.00 (m, 5H, ethyl CH2, methine), 3.09 (m, IH, cyclopentyl methine), 2.96 (m, IH, ArCH2), 2.83 (m, IH, ArCH2), 2.29 (t, 2H, J=7.9, glu 4-CH2), 2.02-1.53 (m, 8H, glu 3- CH2, cyclopentyl CH2), 1.46 (m, 2H, cyclopentyl CH2), 1.15 (t, 3H, J=7.1, ethyl CH3), 1.04 (t, 3H, J=7.1, ethyl CH3). Example 173: Ethyl 5-O-ethyl-L-glutam-l-yl-2-cyclopentyl-L-tyrosinate According to example 48, 0.24 g of ethyl N-((benzyloxy)carbonyl)-5-O-ethyl-L-glutam- l-yl-2-cyclopentyl-L-tyrosinate was hydrogenolyzed using 0.042 g of 10% Pd(C) in 85 mL of MeOH. The crude product was purified by flash chromatography (Siθ2, 95:5 CH2C_2:MeOH) to afford 0.154 g (84%) of ethyl 5-O-ethyl-L-glutam-l-yl-2- cyclopentyl-L-tyrosinate as a thick transparent oil. *H-ΝMR: (300 MHz, DMSO-dό) δ 9.08 (s, IH, OH), 8.23 (d, IH, J=7.9, NH), 6.87 (d, IH, J=8.1, aromatic CH), 6.63 (d, IH, J=2.1, aromatic CH), 6.44 (dd; IH; J=8.1, 2.1; aromatic CH), 4.31 (m, IH, methine), 4.00 (m, 4H, ethyl CH2), 3.12 (m, 2H, glu methine, cyclopentyl methine), 2.98 (m, IH, A1-CH2), 2.82 (m, IH, ArCH2), 2.27 (t, 2H, J=7.9, glu 4-CH2), 2.00-1.35 (m, 12H, glu 3-CH2, NH2, cyclopentyl CH2), 1.07 (t, 3H, J=7.1, ethyl CH3), 1.16 (t, 3H, J=7.1, ethyl CH3). Example 174: Ethyl N*^4-fff2.4-di_-mino-6-pteridinyl)methvπmethylamino benzoylV5- O-ethyl-L-glutam- 1 -yl-2-cvclopentyl-L-tyrosinate According to example 49, 0.131 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate was coupled with 0.15 g of ethyl 5-O-ethyl-L-glutam-l-yl-2-cyclopentyl-L-tyrosinate using 0.08 mL of DECP and 0.10 mL of Et3Ν in 12 mL of DMF. The crude product was subjected to flash chromatography twice on silica gel (93:7 CH2Cl2:MeΟH; 92:8 CH2Cl2:MeΟH ) to afford 0.134 g (52%) of ethyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-ethyl-L-glutam-l-yl-2-cyclopentyl-L- tyrosinate as a yellow powder, m.p. 135-150°C. JH-ΝMR: (300 MHz, DMSO-dό) δ 9.06 (s, IH, OH), 8.54 (s, IH, pteridinyl 7-CH), 8.34 (d, IH, J=7.4, amide ΝH), 7.96 (d, IH, J=8.2, amide ΝH), 7.70 (d, 2H, J=8.6, aromatic CH), 7.63 (br s, IH, ΝH2), 7.43 (br s, IH, NH2), 6.86 (d, IH, J=8.4, aromatic CH), 6.80 (d, 2H, J=8.9, aromatic CH), 6.60 (m, 3H, aromatic CH, NH2), 6.40 (dd; IH; J=8.2, 1.9; aromatic CH), 4.77 (s, 2H, pteridinyl-CH2), 4.42 (m, IH, methine), 4.25 (m, IH, methine), 4.07-3.89 (m, 4H, ethyl CH2), 3.19 (s, 3H, N-CH3), 3.08 (m, IH, cyclopentyl methine), 2.94 (m, IH, ArCH2), 2.82 (m, IH, ArCH2), 2.31 (t, 2H, J=7.9, glu 4-CH2), 2.04-1.32 (m, 10H, glu 3-CH2, cyclopentyl CH2), 1.13 (t, 3H, J=7.1, ethyl CH3), 1.02 (t, 3H, J=7.1, ethyl CH3). Example 175 : N-f 4-f f f2.4-Diamino-6-pteridinyDmethvfimethylan_ύno)beιτzoyl'.-L- glutam- 1 -yl-2-cvclopentyl-L-tyrosine A solution of 0.130 g of ethyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)memylamino)benzoyl)-5-O-ethyl-L-glutam-l-yl-2-cyclopentyl-L- tyrosinate in 10 mL of 1 : 1 THF:H2θ was treated with 17 mg of LiOH and the solution was stirred at RT under nitrogen. The hydrolysis was monitored by HPLC (C18, 70:30:0.1 H2θ:MeCΝ:TFA, flow rate=l mlJmin). An additional 10 mg portion of LiOH was added after 3h. Analysis by HPLC (above conditions) 2h after the second LiOH addition indicated a single component at k'=0.62. The solution was neutralized by addition of IN HCl. A yellow precipitate resulted which was collected by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, frozen, and lyophilized to afford 0.106 g (83%) of N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-2-cyclopentyl-L-tyrosine as a yellow powder, m.p. 185°C (dec). HPLC: one peak on C18, k'=2.10, 75:25:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. Ϊ H-NMR: (300 MHz, DMSO-dό) δ 12.75-11.85 (br m, COOH), 9.03 (br s, IH, OH), 8.56 (s, IH, pteridinyl 7-CH), 8.13 (d, IH, J=7.9, amide NH), 7.96 (d, IH, J=7.8, amide NH), 7.72 (br s, IH, NH2), 7.70 (d, 2H, J=8.7, aromatic CH), 7.53 (br s, IH, NH 2 ), 6.89 (d, IH, J=8.4, aromatic CH), 6.80 (m, 3H, aromatic CH, NH 2 ), 6.69 (br s, IH, H ), 6.60 (s, IH, aromatic CH), 6.38 (dd; IH; J=8.3, 2.2; aromatic CH), 4.78 (s, 2H, pteridinyl-CH 2 ), 4.41 (m, IH, methine), 4.22 (m, IH, methine), 3.21 (s, 3H, N-CH3), 3.12 (m, IH, cyclopentyl methine), 3.00 (dd; IH; J=14.3, 5.3; ArCH2), 2.76 (dd; IH; J=14.3, 8.8; ArCH2), 2.23 (t, 2H, J=7.9, glu 4-CH2), 2.05-1.30 (m, 10H, glu 3-CH2, cyclopentyl CH2). Elemental Analysis: Calcd. for C34H39N9O7 2.5 H2O (MW 730.78): C, 55.88; H, 6.07; N, 17.25. Found: C, 55.91; H, 5.94; N, 17.10. Mass Spectrum: (Ion Spray) 686 (M+H) + . UN Spectrum: (pH 7 Buffer) λmax (ε) 259.0 (25000), 308.2 (25500), 371.5 (7960). λmin (ε) 242.3 (16000), 273.0 (17700), 346.8 (6350). sh (e) 220.9 (30100), 288.1 (21000). Example 176: tert-Butyl 3.5-diiodo-L-tyrosinate A solution of 5.0 g of 3,5-diiodo-L-tyrosine and 0.95 mL of 70% aqueous HCIO4 in 155 mL of tert-butyl acetate was stirred at RT under nitrogen for 4 days. The solution was neutralized by addition of 150 mL of 5% aqueous ΝaHCθ3 and the mixture was subjected to rotary evaporation to remove tert-butyl acetate. An off-white precipitate resulted which was collected by filtration and dried in vacuo. The crude product was subjected to flash chromatography on silica gel (97:3 CH2Cl2:MeOH) to afford 4.2 g (75%) of tert-butyl 3,5-diiodo-L-tyrosinate as a white powder, m.p. 165-168°C. Example 177: tert-Butyl N-f9-fluorenylmethyloxycarbonyl)-5-O-tert-butyl-L-glutam-l- yl-3.5-diiodo-L-tyrosinate According to example 145, 1.50 g of tert-butyl 3,5-diiodo-L-tyrosinate was coupled with 1.31 g of N-FMOC-L-glutamic acid γ-tert-butyl ester using 0.62 g of EDC in 45 mL of CH2CI2 for 3h. The crude product was purified by flash chromatography on silica gel (7:3 hexane:EtOAc) to afford 1.78 g (65%) of tert-butyl N-(9- fluorenylmethyloxycarbonyl)-5-O-tert-butyl-L-glutam-l-yl-3,5-diiodo-L-tyrosinate as a light brown foam. -1H-ΝMR: (200 MHz, DMSO-dό) δ 9.41 (s, IH, OH), 8.27 (d, IH, J=7.0, ΝH), 7.91 (d, 2H, J=7.2, aromatic CH), 7.75 (m, 2H, aromatic CH), 7.61 (s, 2H, aromatic CH), 7.57-7.28 (m, 5H, aromatic CH, ΝH), 4.26 (m, 4H, fluorenyl-CH2θ, fluorenyl methine, α-methine), 4.08 (m, IH, α-methine), 2.83 (d, 2H, J=7.4, ArCH2), 2.25 (t, 2H, J=7.9, glu 4-CH2), 1.83 (br m, 2H, glu 3-CH2), 1.41 (s, 9H, t-Bu), 1.33 (s, 9H, t-Bu). Example 178: tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-3.5-diiodo-L-tyrosinate To a solution of 1.66 g of tert-butyl N-(9-fluorenylmethyloxycarbonyl)-5-O-tert-butyl-L- glutam-l-yl-3,5-diiodo-L-tyrosinate in 7 mL of anhydrous DMF were added 1.71 mL of piperidine (Sigma). After stirring at RT under nitrogen for lh, the solution was mixed with 75 mL of water and was extracted with CH2CI2. The combined CH2CI2 solutions were washed with aqueous ΝaCl (4x30 mL), dried over MgSθ4, and concentrated in vacuo to give a white solid. Analysis of the crude product by tic (Siθ2, 95:5 CH2Cl2:MeΟH) indicated major new components at Rf=04 and 0.5. The Rf=0.4 material was isolated by flash chromatography on silica gel (95:5 CH2C12:MeΟH) to afford 1.1 g (87%) of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3,5-diiodo-L-tyrosinate as a white foam, m.p. 45-57°C. A sample of the corresponding HCl salt was prepared for ΝMR by treating 10 mg of product with IN ethereal HCl and concentrating to dryness. JH-ΝMR: (200 MHz, DMSO-dό) δ 9.42 (br s, IH, OH), 8.98 (d, IH, J=7.0, ΝH), 8.33 (br s, 3H, ΝH3 + ), 7.67 (s, 2H, aromatic CH), 4.36 (m, IH, methine), 3.88 (m, IH, methine), 2.86 (d, 2H, J=7.0, ArCH2), 2.38 (m, 2H, glu 4-CH2), 2.01 (m, 2H, glu 3- CH2), 1.41 (s, 9H, t-Bu), 1.35 (s, 9H, t-Bu). Example 179: tert-Butyl N44-((( 2.4-diamino-6- tyrosinate According to example 154, 0.25 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3,5-diiodo- L-tyrosinate was coupled with 0.132 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)-N- methylamino)benzoic acid hemihydrochloride dihydrate using 0.06 mL of DECP, 0.07 mL of Et3N, and 10 mL of DMF. The crude product was purified by flash chromatography on silica gel (95:5 CH2Cl2:MeOH) to afford 0.18 g (50%) of tert-butyl Λ^4^((2,4*κtt__mino-6-pteridinyl)methy l-yl-3,5-dϋodo-L-tyrosinate as a yellow powder, m.p. 172°C (dec). Example 180: N-f4-f f f 2.4-Diamino-6-pteridinyl'.methyl')methylan_ino)benzoyl)-L- glutam- 1 -yl-3.5-diiodo-L-tyrosine According to example 50, 0.119 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3,5-diiodo-L- tyrosinate was treated with gaseous HCl in 15 mL of CH3ΝO2 for 30 min. The yellow suspension was concentrated in vacuo to dryness. Analysis of the product by ^H-NMR and reverse phase HPLC indicated the desired product contaminated with minor impurities. The mixture was purified by semi-preparative HPLC (C18, 77:23:0.1 H2θ:MeCN:TFA). Fractions containing pure product were combined and subjected to rotary evaporation to remove MeCN. The resulting suspension was frozen and lyophilized to afford 0.024 g (18%) of N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3,5-diiodo-L-tyrosine as a yellow powder, m.p. 205°C (dec). HPLC: one peak on C18, k'=2.74, 77:23:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. -1H-NMR: (300 MHz, DMSO-dό) δ 13.20-11.90 (br m, COOH), 9.35 (s, IH, OH), 9.21 (br s, IH, NH2), 9.02 (br s, IH, NH2), 8.70 (s, IH, pteridinyl 7-CH), 8.52 (br s, IH, NH2), 8.13 (d, IH, J=7.6, amide NH), 7.98 (d, IH, J=7.9, amide NH), 7.72 (d, 2H, J=9.0, aromatic CH), 7.57 (s, 2H, aromatic CH), 7.51 (br s, IH, NH2), 6.80 (d, 2H, J=8.9, aromatic CH), 4.86 (s, 2H, pteridinyl-CH2), 4.41 (m, IH, methine), 4.30 (m, IH, methine), 3.23 (s, 3H, N-CH3), 2.81 (m, 2H, ArCH2), 2.25 (t, 2H, J=7.6, glu 4-CH2 ), 1.90 (m, 2H, glu 3-CH2). Elemental Analysis: Calcd. for C29H29N9O7I2 I 7 TFA-2.3 H2O (MW 1104.69): C, 35.23; H, 3.22; N, 11.41. Found: C, 35.25; H, 3.24; N, 11.44. Mass Spectrum: (Ion Spray) 870 (M+H) + . UN Spectrum: (pH 7 Buffer) λ ma χ (ε) 219.7 (48900), 258.0 (26500), 306.0 (26200), 373.3 (7780). λmin (ε) 214.9 (48300), 246.0 (23700), 273.7 (18400), 347.6 (6200). Example 181: Methyl 4-hvdroxy-3-iodobenzoate To a 500 mL 3-necked flask equipped with a magnetic stirrer, a thermometer, and a condenser were added 10.0 g of methyl 4-hydroxybenzoate (Aldrich) and 30 mL of glacial acetic acid. To this suspension was added a solution of 11.74 g of iodine monochloride in 30 mL of glacial acetic acid via addition funnel. The solution was heated at 80°C for lh, 45°C for 18h, and 90°C for an additional 4h. Cooling to RT afforded a thick red-orange suspension which was transferred to a IL separatory funnel and dissolved in 500 mL of CHCI3 by shaking vigorously. The dark puφle solution was washed with water (2x100 mL), saturated aqueous sodium thiosulfate (2x100 mL), and 5% aqueous ΝaHCθ3 (2x100 mL). A light yellow solution resulted which was dried over MgSO4, and concentrated in vacuo to give a white solid. This material was recystallized from water-MeOH to afford 14.92 g (82%) of methyl 4-hydroxy-3- iodobenzoate as a white crystalline solid, m.p. 145-146°C. Example 182: Methyl 4-hvdroxy-3-cvclopentylbenzoate According to example 151, 5.0 g of methyl 4-hydroxy-3-iodobenzoate was subjected to Heck coupling with 25 mL of cyclopentene using 80 mL of toluene, 0.40 g of Pd(OAc)2, 1.10 g of tri-ortho-tolylphosphine, and 2.76 mL of Et3N. The reaction was carried out at 110°C for 24h. Analysis of the crude product by tic (Siθ2, 75:25 hexane:EtOAc) indicated a major new component at Rf*=0.50. This material was isolated by flash chromatography on silica gel (75:25 hexane:EtOAc). This gave 2.97 g (76%) of the desired olefin mixture (1H-NMR) as a brown solid. This material was subjected to catalytic hydrogenation at 45 psi for 18h using 80 mL of MeOH and 1.0 g of 10% Pd(C). This afforded 2.59 g (65% for both steps) of methyl 4-hydroxy-3-cyclopentylbenzoate as a white solid, m.p. 114-116°C. Example 183: Methyl 4-fftert-butyldimethylsily0oxyV3-cyclopentylbenzoate A 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet was charged with 1.79 g of methyl 4-hydroxy-3-cyclopentylbenzoate, 1.22 g of imidazole (Aldrich), 30 mL of anhydrous DMF, and 1.47 g of tert-butyldimethylsilyl chloride (Aldrich). The solution was stirred at RT under nitrogen for 24h and then concentrated in vacuo to give a thick oil. This material was subjected to flash chromatography on silica gel (95:5 hexane:EtOAc) to afford 2.43 g (89%) of methyl 4-((tert- butyldimethylsilyl)oxy)-3-cyclopentylbenzoate as a clear liquid. JH-NMR: (200 MHz, DMSO-dό) δ 7.81 (d, IH, J=2.0, aromatic CH), 7.72 (dd; IH; J=8.4, 2.0; aromatic CH), 6.93 (d, IH, J=8.4, aromatic CH), 3.80 (s, 3H, CH3), 3.30 (m, IH, cyclopentyl methine), 2.06-1.37 (m, 8H, cyclopentyl CH2), 0.98 (s, 9H, t-Bu), 0.27 (s, 6H, SiMe2). Example 184: 4-fftert-Butyldimethylsilyl')oxyV3-cyclopentylbenzyl alcohol To a 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 2.58 g of methyl 4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylbenzoate and 15 mL of anhydrous toluene. The solution was cooled in an ice water bath and was treated with 19.3 mL of IM diisobutylaluminum hydride in toluene (Aldrich). The solution was stirred at 0°C for 30 min and was allowed to warm to RT. After 2h at RT the solution was poured into 130 mL of saturated aqueous potassium sodium tartrate and the resulting mixture was stirred for 20 min. The mixture was then extracted with ethyl ether (3x70 mL). The combined ether extracts were washed with water (3x70 mL), dried over anhydrous MgSO4, and concentrated to give a cloudy oil. The crude product was purified by flash chromatography on silica gel (8:2 hexane:EtOAc) to afford 1.94 g (82%) of 4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylbenzyl alcohol as a clear liquid. JH-NMR: (200 MHz, DMSO-dό) δ 7.18 (d, IH, J=2.0, aromatic CH), 7.02 (dd; IH; J=8.2, 2.2; aromatic CH), 6.76 (d, IH, J=8.0, aromatic CH), 5.02 (t, IH, J=5.8, OH), 4.40 (d, 2H, J=5.6, ArCH2θ), 3.29 (m, IH, cyclopentyl methine), 2.04-1.38 (m, 8H, cyclopentyl CH2), 1.03 (s, 9H, t-Bu), 0.22 (s, 6H, SiMe2). Example 185: 4- tert-Butyldimethylsilyl'.oxy'.-3-cvclopentylbenzaldehvde A solution of 1.94 g of 4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylbenzyl alcohol in 70 mL of anhydrous CH2CI2 was treated with 2.05 g of pyridinium chlorochromate (Aldrich) and was stirred at RT under nitrogen. After 30 min, tic (Siθ2, 85:15 hexane.EtOAc) indicated no remaining starting material and a major new component at Rf=0.80. The mixture was filtered to remove solids and the filtrate was washed with 5% aqueous citric acid (4x50 mL) followed by 5% aqueous NaHCO3 (4x50 mL). The CH2CI2 solution was dried over anhydrous MgSO4 and concentrated in vacuo to give a brown oil. This material was purified by flash chromatography on silica gel (9: 1 hexane:EtOAc) to afford 1.52 g (79%) of 4-((tert-butyldimethylsilyl)oxy)-3- cyclopentylbenzaldehyde as a clear oil. JH-NMR: (200 MHz, DMSO-d6) δ 9.88 (s, IH, aldehyde CH), 7.80 (d, IH, J=2.0, aromatic CH), 7.69 (dd; IH; J=8.2, 2.0; aromatic CH), 7.03 (d, IH, J=8.3, aromatic CH), 3.29 (m, IH, cyclopentyl methine), 2.08-1.43 (m, 8H, cyclopentyl CH2), 1.01 (s, 9H, t-Bu), 0.30 (s, 6H, SiMe2). Example 186: tert-Butyl trans-4-( ftert-butyldimethylsilyπoxyV3-cyclopentylcinnamate To a 100 mL 3-necked flask equipped with a magnetic stirrer, a nitrogen inlet and a reflux condenser were added 1.57 g of tert-butyl diethylphosphonoacetate (Aldrich) and 20 mL of anhydrous ethyl ether. This was followed by addition of 0.40 g of 60% NaH mineral oil dispersion. The resulting cloudy solution was heated at reflux for 25 min, cooled to 0°C and treated with a solution of 1.52 g of 4-((tert-butyldimethylsilyl)oxy)-3- cyclopentylbenzaldehyde in 10 mL of ether. A thick yellow gel resulted which was broken up with a spatula. The mixture was allowed to warm to RT and stirring was continued for an additional lh. Analysis by tic (Siθ2, 95:5 hexane:EtOAc) indicated a major new component at R =0.48 and minor components at Rf=0.06, 0.41 and 0.98. The mixture was diluted with 70 mL of ether, washed with saturated brine, and dried over anhydrous MgSO4. The drying agent was removed by filtration and the filtrate concentrated to give a yellow oil. This material was purified by flash chromatography on silica gel (97:3 hexane:EtOAc) to afford 1.53 g (77%) of tert-butyl trans-4-((tert- butyldimethylsilyl)oxy)-3-cyclopentylcinnamate as a clear oil. Ϊ H NMR: (200 MHz, DMSO-dό) δ 7.59-7.36 (m, 3H, aromatic CH, olefinic CH), 6.83 (d, IH, J=8.4, aromatic CH), 6.38 (d, IH, J=15.8, olefinic CH), 3.27 (m, IH, cyclopentyl methine), 2.02-1.52 (m, 8H, cyclopentyl CH2), 1.49 (s, 9H, t-Bu), 1.00 (s, 9H, Si-t-Bu), 0.25 (s, 6H, SiMe2). Example 187: f2R. 3S.-tert-Butyl 3-f4-fftert-butyldimethylsilvnoxyV3- cyclopentylphenylV2.3-dihvdroxypropionate To a 250 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 20 mL of tert-butyl alcohol, 20 mL of water, 5.32 g of AD-mix-α (Shaφless, K.B.; et al. J. Org. Chem., 1992, 57, 2768-2771; reagent purchased from Aldrich) and 0.36 g of methanesulfonamide (Aldrich). This was followed by addition of 1.53 g of tert-butyl _>ταtιs-4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylcinnamate. The reaction mixture was stirred at RT under nitrogen. After 18h, 5.7 g of sodium sulfite were added and the mixture was stirred for 30 min. The mixture was concentrated in vacuo to 15 mL, diluted with 100 mL of water and extracted with EtOAc (4x50 mL). The combined EtOAc extracts were washed with water (3x50 mL), dried over anhydrous MgSO4 and concentrated to give a cloudy oil. This material was purified by flash chromatography on silica gel (75:25 hexane:EtOAc) to afford 1.44 g (87%) of tert-butyl (2R, 3S)-3-(4-((tert- butyldimethylsilyl)oxy)-3-cyclopentylphenyl)-2,3-dihydroxypropionate as a thick clear oil. JH-NMR: (200 MHz, DMSO-dό) δ 7.16 (s, IH, aromatic CH), 7.01 (d, IH, J=8.2, aromatic CH), 6.73 (d, IH, J=8.2, aromatic CH), 5.28 (d, IH, J=5.4, OH), 5.25 (d, IH, J=7.1, OH), 4.55 (t, IH, J=5.6, propionate 2-methine), 3.93 (t, IH, J=6.8, propionate 3- methine), 3.27 (m, IH, cyclopentyl methine), 2.04-1.37 (m, 8H, cyclopentyl CH2), 1.24 (s, 9H, t-Bu), 0.99 (s, 9H, Si-t-Bu), 0.21 (s, 6H, SiMe2). Example 188: (R ert-Butyl 3-f4-fftert-butyldimethylsilvftoxyV3-cvclopentylphenyl'.-2- hvdroxypropionate To a 300 mL Parr bottle were added 0.35 g of 10% Pd(C) and 20 mL of tert-butyl alcohol under a nitrogen flush. To this was added a solution of 1.42 g of tert-butyl (2R, 3S)-3-(4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylphenyl)-2,3-dihydroxypropionate in 40 mL of tert-butyl alcohol. The mixture was deoxygenated by bubbling nitrogen through for 5 min while gently warming the bottle to avoid freezing of tert-butyl alcohol. The mixture was treated with 4 drops of concentrated HCIO4 and hydrogenated at 45 psi and 40°C for 72h. The vessel was purged with nitrogen, catalyst removed by filtration through celite, and the filtrate concentrated in vacuo to 20 mL. The solution was mixed with 60 mL of 5% aqueous NaHCO3 and the mixture extracted with EtOAc (4x40 mL). The combined EtOAc extracts were washed with 5% aqueous NaHCO3 (3x50 mL), dried over anhydrous MgSO4 and concentrated to give a cloudy oil. This material was purified by flash chromatography on silica gel (9:1 hexane:EtOAc) to afford 1.17 g (86%) of tert-butyl (R)-3-(4-((tert-butyldimethylsilyl)oxy)-3-cycloρentylphenyl)-2- hydroxypropionate as a thick transparent oil. IH-NMR: (200 MHz, DMSO-dό) δ 7.05 (d, IH, J=2.1, aromatic CH), 6.92 (dd; IH; J=8.4, 2.1; aromatic CH), 6.69 (d, IH, J=8.2, aromatic CH), 5.33 (d, IH, J=5.7, OH), 4.08 (m, IH, methine), 3.23 (m, IH, cyclopentyl methine), 2.78 (d, 2H, J=6.1, ArCH2), 2.00-1.38 (m, 8H, cyclopentyl CH2), 1.32 (s, 9H, t-Bu), 0.99 (s, 9H, Si-t-Bu), 0.20 (s, 6H, SiMe2). Example 189: fS ert-Butyl 2-ffN-ffbenzyloxy.carbonvn-5-O-tert-butyl-L-glutam-l- yπoxyV3-f4-fftert-butyldimethylsilyπoxyV3-cyclopentylphenvπpropionate To a 100 mL 3-necked flask equipped with a magnetic stirrer and a nitrogen inlet were added 1.14 g of tert-butyl (R)-3-(4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylphenyl)-2- hydroxypropionate, 20 mL of anhydrous THF, 1.01 g of N-Cbz-L-glutamic acid γ-tert- butyl ester (Sigma), and 0.78 g of triphenylphosphine (Aldrich). The solution was cooled to 0°C and was treated with 0.47 mL of diethyl azodicarboxylate (Aldrich) by dropwise addition. The solution was allowed to warm to RT and stirring under nitrogen was continued. After 3.5h, tic (Siθ2, 85:15 hexane:EtOAc) indicated a major new component at Rf=0.46 and some remaining alcohol starting material at Rf=0.51. The solution was again cooled to 0°C and treated with an additional 0.18 g of N-Cbz-L- glutamic acid γ-tert-butyl ester, 0.14 g of triphenylphosphine, and 0.085 mL of diethyl azodicarboxylate. After warming to RT and stirring for 18h tic indicated only a trace of starting material. The solution was concentrated in vacuo to dryness and the crude product purified by flash chromatography (Siθ2, 85:15 hexane:EtOAc). This afforded 1.86 g (84%) of (S)-tert-butyl 2-((N-((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-l- yl)oxy)-3-(4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylphenyl)propionate as a thick transparent oil. *H-ΝMR: (200 MHz, DMSO-dό) δ 7.76 (d, IH, J=8.0, ΝH), 7.34 (m, 5H, aromatic CH), 7.11 (s, IH, aromatic CH), 6.96 (d, IH, J=8.1, aromatic CH), 6.68 (d, IH, J=8.1, aromatic CH), 5.01 (m, 3H, PhCH2θ, propionate methine), 4.19 (m, IH, glu methine), 3.21 (m, IH, cyclopentyl methine), 3.01 (d, 2H, J=5.1, ArCH2), 2.32 (t, 2H, J=7.8, glu 4-CH2), 2.12-1.43 (m, 10H, glu 3-CH2, cyclopentyl CH2), 1.39 (s, 9H, t-Bu), 1.26 (s, 9H, t-Bu), 0.98 (s, 9H, Si-t-Bu), 0.19 (s, 9H, SiMe2). Example 190: fS tert-Butyl 2-ffN-ffbenzyloxy'.carbonyl > .-5-O-tert-butyl-L-glutam-l- vπoxyV3-f3-cvclopentyl-4-hvdroxyphenyπpropionate A solution of 1.85 g of (S)-tert-butyl 2-((N-((benzyloxy)carbonyl)-5-O-tert-butyl-L- glutam- 1 -yl)oxy)-3-(4-((tert-butyldimethylsilyl)oxy)-3-cyclopentylphenyl)propionate in 20 mL of anhydrous THF was treated with 2.63 mL of IM tetrabutylammonium fluoride/THF (Aldrich) and was stirred at 0°C under nitrogen. After 15 min, tic (Siθ2, 75:25 hexane:EtOAc) indicated no remaining starting material and a single new component at Rf=0.41. The solution was mixed with 80 mL of water and was extracted with EtOAc (4x40 mL). The combined EtOAc extracts were washed with saturated brine (3x60 mL), dried over anhydrous MgSO4 and concentrated in vacuo to give a clear oil. This material was subjected to flash chromatography on silica gel (75:25 hexane:EtOAc) to afford 1.5 g (96%) of (S)-tert-butyl 2-((N-((benzyloxy)carbonyl)-5-O- tert-butyl-L-glutam-l-yl)oxy)-3-(3-cyclopentyl-4-hydroxyphenyl)propionate as a white foam. H-ΝMR: (200 MHz, DMSO-dό) δ 9.11 (s, IH, OH), 7.77 (d, IH, J=8.2, NH), 7.33 (m, 5H, aromatic CH), 7.01 (d, IH, J=1.6, aromatic CH), 8.85 (dd; IH; J=8.2, 1.7; aromatic CH), 6.68 (d, IH, J=8.2, aromatic CH), 5.04 (s, 2H, PI1CH2O), 4.97 (m, IH, propionate methine), 4.18 (m, IH, glu methine), 3.17 (m, IH, cyclopentyl methine), 2.96 (d, 2H, J=6.0, ArCH2), 2.34 (t, 2H, J=7.4, glu 4-CH2), 2.13-1.43 (m, 10H, glu 3-CH2, cyclopentyl CH2), 1.40 (s, 9H, t-Bu), 1.29 (s, 9H, t-Bu). Example 191: fSVtert-Butyl 2-ff5-O-tert-butyl-L-dutam-l-vnoxyV3-f3-cvclopentyl-4- hvdroxyphenyπpropionate According to example 48, 1.49 g of (S)-tert-butyl 2-((N-((benzyloxy)carbonyl)-5-O-tert- butyl-L-glutam- 1 -yl)oxy)-3-(3-cyclopentyl-4-hydroxyphenyl)propionate was hydrogenolyzed using 0.24 g of 10% Pd(C) in 70 mL of MeOH. Analysis of the crude product by tic (Siθ2, 4:6 hexane.EtOAc) indicated a major component at Rf= 0.47 and minor components at Rf=0.60 and 0.15. The crude product was purified by flash chromatography on silica gel (4:6 hexane:EtOAc) to afford 0.76 g (65%) of (S)-tert- butyl 2-((5-O-tert-butyl-L-glutam-l-yl)oxy)-3-(3-cyclopentyl-4- hydroxyphenyl)propionate as a white foam. JH-ΝMR: (200 MHz, DMSO-dό) δ 9.12 (s, IH, OH), 7.00 (s, IH, aromatic CH), 6.86 (d, IH, J=8.2, aromatic CH), 6.68 (d, IH, J=8.2, aromatic CH), 4.92 (t, IH, J=6.2, propionate methine), 4.04 (q, IH, J=7.0, glu methine), 3.19 (m, IH, cyclopentyl methine), 2.95 (d, 2H, J=6.4, ArCH2), 2.33 (t, 2H, J=7.4, glu 4-CH2), 2.00-1.48 (m, 12H, glu 3-CH2, ΝH2, cyclopentyl CH2), 1.39 (s, 9H, t-Bu), 1.31 (s, 9H, t-Bu). Example 192: (S) -tert-Butyl 3-f3-cvclopentyl-4-hvdroχyphenylV2-ff5-O-tert-butyl- 4- fff2.4-diamino-6-pteridinyl ' )methvπmethylamino)benzoylVL-glutam-l-yl ' )oxy ' )propionate According to example 154, 0.414 g of (S)-tert-butyl 2-((5-O-tert-butyl-L-glutam-l- yl)oxy)-3-(3-cyclopentyl-4-hydroxyphenyl)propionate was coupled with 0.32 g of 4-(N- (2,4-diamino-6-pteridinylmethyl)-N-methylamino)benzoic acid hemihydrochloride dihydrate using 0.19 mL of DECP, 0.23 mL of Et3N, and 25 mL of DMF. The crude product was subjected to flash chromatography twice on silica gel (14: 14: 1 CH2Cl2:acetone:MeOH; 95:5_€91:9 CH2Cl2:MeOH) to afford 0.46 g (69%) of (S)-/ert- butyl 3*^3*^clopentyl-4-hydroxvphenyl)-2-((5-O-tert-butyH4-(((2,4-di_ιmino-6- pteridinyl)methyl)methylanMno)benzoyl)-L-glutam-l-yl)oxy)propionate as a yellow powder, m.p. 135-137°C. JH-NMR: (300 MHz, DMSO-dό) δ 9.07 (s, IH, OH), 8.55 (s, IH, pteridinyl 7-CH), 8.30 (d, IH, J=7.6, amide NH), 7.71 (d, 2H, J=8.6, aromatic CH), 7.64 (br s, IH, NH2), 7.42 (br s, IH, NH2), 6.97 (d, IH, J=1.6, aromatic CH), 6.80 (m, 3H, aromatic CH), 6.59 (m, 3H, aromatic CH, NH2), 4.92 (t, IH, J=6.1, propionate methine), 4.77 (s, 2H, pteridinyl-CH 2 ), 4.49 (m, IH, glu methine), 3.19 (s, 3H, N-CH3), 3.12 (m, IH, cyclopentyl methine), 2.93 (m, 2H, A1-CH2), 2.33 (t, 2H, J=7.2, glu 4-CH2), 2.08 (m, IH, glu 3-CH2), 1.87 (m, 3H, glu 3-CH2, cyclopentyl CH2), 1.77-1.40 (m, 6H, cyclopentyl CH2), 1.36 (s, 9H, t-Bu), 1.25 (s, 9H, t-Bu). Example 193 : fS. -3-f 3-Cvclopentyl-4-hvdroxyphenvn-2- (N 4- f 2.4-diamino-6- pteridinyl'.methyl'.methylamino'.benzoyπ-L-glutam- 1 -yπoxy'.propionic acid According to example 50, 0.43 g of (SVtert-butyl 3-(3-cyclopentyl-4-hydroxyphenyl)-2- ((5-O-tert-butyl-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- l-yl)oxy)propionate was treated with gaseous HCl in 50 mL of CH3NΟ2 for 45 min. The yellow suspension was concentrated in vacuo to dryness. The product was suspended in 25 mL of water and was treated with sufficient IN aqueous NaOH to give complete solution. The solution was filtered and the pH adjusted to 5.5 by addition of IN HCl. A yellow precipitate resulted which was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, frozen and lyophilized to afford 0.345 g (88%) of (S)-3-(3-cyclopentyl-4-hydroxyphenyl)-2-((N-(4- (((2,4-diammo-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl)oxy)propionic acid as a yellow-orange powder, m.p. 165°C (dec). HPLC: one major peak on C18, k'=2.02; 3.6% of methotrexate was detected at k'=0.09; 70:30:0.1 H2θ:MeCN:TFA, flow rate=l mlJmin. *H-NMR: (300 MHz, DMSO-dό) δ 12.40-11.90 (br s, COOH), 9.03 (br s, IH, OH), 8.57 (s, IH, pteridinyl 7-CH), 8.27 (d, IH, J=7.6, amide NH), 7.86 (br s, IH, NH2), 7.70 (d, 2H, J=8.9, aromatic CH), 7.65 (br s, IH, NH2), 6.97 (d, IH, J=1.6, aromatic CH), 6.80 (m, 5H, NH2, aromatic CH), 6.61 (d, IH, J=8.2, aromatic CH), 4.96 (t, IH, J=5.9, methine), 4.78 (s, 2H, pteridinyl-CH 2 ), 4.49 (m, IH, methine), 3.19 (s, 3H, N- CH3), 3.16 (m, IH, cyclopentyl methine), 2.94 (d, 2H, J=5.8, ArCH 2 ), 2.34 (t, 2H, J=7.5, glu 4-CH 2 ), 2.10 (m, IH, glu 3-CH 2 ), 1.87 (m, 3H, glu 3-CH 2 , cyclopentyl CH 2 ), 1.78-1.40 (m, 6H, cyclopentyl CH2). Elemental Analysis: Calcd. for C34H38N8O8 2.1 H2O (MW 724.56): C, 56.36; H, 5.87; N, 15.47. Found: C, 56.13; H, 5.59; N, 15.46. Mass Spectrum: (Ion Spray) 687 (M+H) + . UN Spectrum: (pH 7 Buffer) λmax (ε) 259.2 (23100), 307.3 (23300), 372.4 (7190). λmin (ε) 242.0 (14300), 272.9 (16900), 343.6 (5460). sh (e) 219.8 (26400), 280.7 (19900). Example 194: Methyl 3-tert-butyl-4-hydroxybenzoate To a 500 mL 3-necked flask equipped with a magnetic stirrer, a nitrogen inlet, and a condenser were added 10.0 g of 3-tert-butyl-4-hydroxybenzoic acid (ICΝ Biomedicals. Inc., Aurora, Ohio 44202), and 200 mL of MeOH. The solution was acidified by bubbling HCl gas through for 5 min. and was then heated at reflux for 5h. The solution was cooled to RT and concentrated in vacuo to dryness. The resulting solid was dissolved in EtOAc. The solution was washed three times with 5% aqueous ΝaHC03, dried over anhydrous MgSO4 and concentrated to dryness to afford 9.65 g (90%) of methyl 3-tert-butyl-4-hydroxybenzoate as a white crystalline solid, m.p. 144-145°C. Example 195: Methyl 3-tert-butyl-4-fftert-butyldimethylsilyl)oxy)benzoate According to example 183, 5.00 g of methyl 3-tert-butyl-4-hydroxybenzoate was silylated using 4.34 g of tert-butyldimethylsilyl chloride, 3.59 g of imidazole, and 70 mL of anhydrous DMF. The crude product was purified by flash chromatography on silica gel (9:1 hexane:EtOAc) to afford 6.1 g (79%) of methyl 3-tert-butyl-4-((tert- butyldimethylsilyl)oxy)benzoate as a clear liquid. 'HM: (200 MHz, DMSO-dό) δ 7.89 (d, IH, J=2.3, aromatic CH), 7.76 (dd; IH; J=8.5, 2.3; aromatic CH), 6.98 (d, IH, J=8.4, aromatic CH), 3.82 (s, 3H, CO2CH3), 1.37 (s, 9H, t-Bu), 1.03 (s, 9H, Si-t-Bu), 0.37 (s, 6H, SiMe2). Example 196: 3-tert-Butyl-4-fftert-butyldimethylsilyl'.oxy'.benzyl alcohol According to example 184, 5.00 g of methyl 3-tert-butyl-4-((tert- butyldimethylsilyl)oxy)benzoate was reduced using 31.8 mL of IM diisobutylaluminum hydride/toluene and 50 mL of anhydrous toluene. Analysis of the reaction mixture by tic after 30 min indicated some remaining starting material. To ensure complete reduction, an additional 5 mL of IM diisobutylaluminum hydride/toluene was added and the reaction was allowed to proceed for an additional 5 min. Work-up in the usual manner afforded 4.11 g (90%) of 3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzyl alcohol as a cloudy oil. The product was used without further purification. 1H-NMR: (200 MHz, DMSO-d6) δ 7.20 (d, IH, J=2.0, aromatic CH), 7.04 (dd; IH; J=8.2, 2.1; aromatic CH), 6.80 (d, IH, J=8.0, aromatic CH), 5.01 (t, IH, J=5.7, OH), 4.39 (d, 2H, ArCH2θ), 1.36 (s, 9H, t-Bu), 1.02 (s, 9H, Si-t-Bu), 0.32 (s, 6H, SiMe2). Example 197: 3-tert-Butyl-4-fftert-butyldimethylsilyl)oxy ' .benzyl bromide A 500 mL round-bottomed flask equipped with a magnetic stirrer was charged with 3.6 g of 3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzyl alcohol and 25 mL of anhydrous CH2CI2. The solution was cooled in an ice water bath and was treated with 5.68 g of dibromotriphenylphosphorane (Aldrich). The reaction mixture was warmed to RT, stirred for 2.5h and then diluted with 100 mL of CH2CI2. The resulting solution was washed with 5% aqueous NaHCO3 (4x60 mL), dried over anhydrous MgSO4 and concentrated to give a white solid. The crude product was purified by flash chromatography on silica gel (95:5 hexane:EtOAc) to afford 3.86 g (88%) of 3-tert- butyl-4-((tert-butyldimethylsilyl)oxy)benzyl bromide as a white crystalline solid, m.p. 54- 55°C. Example 198: tert-Butyl f2R. 3S. 5S.-6-oxo-2.3-diphenyl-5-f3-tert-butyl-4-fftert- butyldimethylsilyl ' .oxy ' .benzyπ-4-moφholinecarboxylate According to example 142, 3.82 g of tert-butyl (2R, 3S)-(-V6-oxo-2,3-diphenyl-4- moφholinecarboxylate was alkylated with 3.86 g of3-tert-butyl-4-((tert- butyldimethylsilyl)oxy)benzyl bromide in 80 mL of anhydrous THF using 11.34 mL of 1 M sodium bis(trimethylsilyl)amide in THF. The crude product was subjected to flash chromatography on silica gel (85:15 hexane:EtOAc) to afford 4.85 g (71%) of tert-butyl (2R, 3S, 5S 6-oxo-2,3-diphenyl-5-(3-tert-butyl-4-((tert-butyldimethylsilyl)oxy)benzyl)- 4-moφholinecarboxylate as a white foam, m.p. 75-83°C. Example 199: f2R. 3S. 5S)-6-Oxo-2.3-diphenyl-5-f3-tert-butyl-4- hydroxybenzyπmoφholine To a 250 mL 3-necked flask equipped with a nitrogen inlet and a magnetic stirrer were added 4.51 g of tert-butyl (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert-butyl-4-((tert- butyldimethylsilyl)oxy)benzyl)-4-moφholinecarboxylate and 50 mL of anhydrous THF. The solution was cooled in an ice water bath, treated with 7.51 mL of IM ι_Bu4NF/THF (Aldrich), and then allowed to warm to RT with stirring under nitrogen. After 1.5h, tic (SiO 2 , 85:15 hexane:EtOAc) indicated no remaining starting material and a single new component at Rf-=0.52. The solution was diluted with 125 mL of EtOAc, washed with water (4x100 mL), dried over MgSO4, and concentrated to give a thick, clear oil. Mass spectral analysis (APCI) indicated the desired intermediate with m/e=538 (M+H+Na) + . The oil was dissolved in 50 mL of CH2CI2 and the solution treated with 15 mL of TFA. After stirring for 40 min, tic (Siθ2, 65:35 hexane:EtOAc) indicated a single new component at R 0.36. The solution was neutralized by addition of saturated aqueous NaHCO3. The mixture was transferred to a separatory funnel and the layers separated. The CH 2 C1 2 solution was washed with additional saturated NaHCO3 (3x50 mL), dried over MgSO4 and concentrated in vacuo to give a clear foam. The crude product was purified by flash chromatography (SiO 2 , 7:3 hexane:EtOAc) to afford 2.66 g (89%) of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert-butyl-4-hydroxybenzyl)moφholine as a white foam, m.p. 76-80°C. Example 200: 3-tert-Butyl-L-tyrosine To a 300 mL Parr bottle were added 0.73 g of PdCl2 and 20 mL of 1 : 1 THF:EtOH. To this was added a solution of 2.43 g of (2R, 3S, 5S)-6-oxo-2,3-diphenyl-5-(3-tert-butyl-4- hydroxybenzyl)moφholine in 80 mL of 1 : 1 THF:EtOH. The mixture was deoxygenated by bubbling nitrogen through for 10 min and was hydrogenated at 45 psi for 18h. The vessel was purged with nitrogen, catalyst removed by filtration, and the filtrate concentrated in vacuo. The residue was partitioned between water and hexane. The aqueous solution was washed with four additional 50 mL portions of hexane, subjected to brief rotary evaporation to remove residual hexane, frozen and lyophilized to afford 1.47 g (92%) of 3-tert-butyl-L-tyrosine HCl as a tan hygroscopic solid. -1H-NMR: (200 MHz, DMSO-dό) δ 9.40 (br s, IH, OH), 8.36 (br s, 3H, NH3 + ), 7.04 (s, IH, aromatic CH), 6.91 (d, IH, J=8.2, aromatic CH), 6.78 (d, IH, J=8.2, aromatic CH), 4.05 (m, IH, methine), 3.02 (d, 2H, J=5.6, ArCH2), 1.34 (s, 9H, t-Bu). Example 201: tert-Butyl 3-tert-butyl-L-tyrosinate To a 250 mL round bottomed flask equipped with a magnetic stirrer were added 30 mL of isobutylene (condensed at -78°C), 1.30 g of 3-tert-butyl-L-tyrosine HCl , 60 mL of 1,4-dioxane, and 0.5 mL of concentrated H SO 2 . The flask was sealed with a rubber septum and wired shut. After stirring at RT for 18h, the flask was vented and the contents poured into a solution of 10 g of NaHCO3 in 150 mL of water. The mixture was subjected to rotary evaporation to remove isobutylene. An emulsion resulted which was extracted with EtOAc (4x40 mL). The combined extracts were washed with 5% aqueous NaHCO3 (3x50 mL), dried over anhydrous MgSO4, and concentrated to give a light yellow foam. Analysis of the product by tic (SiO 2 , 95:5 CH 2 Cl 2 :MeOH) indicated major and minor components at Rp0.45 and 0.49 respectively. The Rf=0.45 product was isolated by flash chromatography on silica gel (95:5 CH 2 Cl :MeOH) to afford 0.61 g (44%) of tert-butyl 3-tert-butyl-L-tyrosinate as a white foam, m.p. 43-52°C. Example 202: tert-Butyl N-ffbenzyloxy ' .carbonylV5-O-tert-butyl-L-glutam-l-yl-3-tert- butyl-L-tyrosinate According to example 145, 0.57 g of tert-butyl 3-tert-butyl-L-tyrosinate was coupled with 0.66 g of N-Cbz-L-glutamic acid γ-tert-butyl ester (Sigma) using 0.39 g of EDC in 20 mL of CH 2 C_2 for 4h. The crude product was purified by flash chromatography on silica gel (65:35 hexane:EtOAc) to afford 0.99 g (83%) of tert-butyl N- ((benzyloxy)carbonyl)-5-O-tert-butyl-L-glutam-l-yl-3-tert-butyl-L-tyrosinate as a white foam, m.p. 62-66°C. Example 203: tert-Butyl 5-O-tert-butyl-L-glutam-l-yl-3-tert-butyl-L-tyrosinate According to example 48, 0.93 g of tert-butyl N-((benzyloxy)carbonyl)-5-O-tert-butyl-L- glutam-l-yl-3-tert-butyl-L-tyrosinate was hydrogenolyzed using 0.15 g of 10% Pd(C) in 70 mL of MeOH for 3.5h. The crude product was subjected to flash chromatography on silica gel (EtOAc) to afford 0.63 g (87%) of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3- tert-butyl-L-tyrosinate as a thick transparent oil. JH-ΝMR: (200 MHz, DMSO-dό) δ 9.17 (s, IH, OH), 8.11 (d, IH, J=7.8, ΝH), 6.96 (d, IH, J=1.7, aromatic CH), 6.84 (dd; IH; J=8.1, 1.9; aromatic CH), 6.68 (d, IH, J=8.0, aromatic CH), 4.33 (m, IH, methine), 3.14 (m, IH, methine), 2.84 (d, 2H, J=7.0, ArCH 2 ), 2.23 (t, 2H, J=7.9, glu 4-CH2), 1.88-1.46 (m, 4H, glu 3-CH2, ΝH2), 1.40 (s, 9H, t-Bu), 1.33 (s, 18H, t-Bu's). Example 204: tert-Butyl N-(4-fff2.4-diamino-6- pteridinyl.methyl ' .methylamino)benzoylV5-O-tert-butyl-L-glutam-l-yl-3-tert-butyl-L- tyrosinate According to example 154, 0.320 g of tert-butyl 5-O-tert-butyl-L-glutam-l-yl-3-tert- butyl-L-tyrosinate was coupled with 0.254 g of 4-(N-(2,4-diamino-6-pteridinylmethyl)- N-methylamino)benzoic acid hemihydrochloride dihydrate using 0.15 mL of DECP, 0.19 mL of Et3Ν, and 20 mL of DMF. The crude product was purified by flash chromatography on silica gel (7:7:1 CH2θ2:acetone:MeOH) to afford 0.32 g (61%) of tert-butyl N-(4-(((2,4-di_ιnιino-6-pteridinyl)methyl)methylaπ_ino)benzoyl)-5-O-tert-butyl- L-glutam-l-yl-3-tert-butyl-L-tyrosinate as a yellow powder, m.p. 125°C (dec). Example 205 : N- 4-f f f 2.4-Di-ιmino-6-pteridinyl)methyDmethylamino ' .benzoyD-L- glutam- 1 -yl-3-tert-butyl-L-tyrosine According to example 50, 0.287 g of tert-butyl N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-5-O-tert-butyl-L-glutam-l-yl-3-tert-butyl-L- tyrosinate was treated with gaseous HCl in 55 mL of CH3ΝO2 for lh. The suspension was concentrated in vacuo to give a yellow powder. This material was suspended in 30 mL of water and treated with sufficient IN aqueous NaOH to give complete solution. The yellow solution was filtered and acidified to pH=5.5 by addition of IN aqueous HCl. The resulting yellow precipitate was separated by centrifugation, washed with four cycles of aqueous suspension-centrifugation-decantation, frozen and lyophilized to afford 0.178 g (68%) of N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 - yl-3-tert-butyl-L-tyrosine as a yellow powder, m.p. 185°C (dec). HPLC: one peak on C18, k'=3.14, 75:25:0.1 H2θ:MeCΝ:TFA, flow rate=l mL/min. -1H-NMR: (300 MHz, DMSO-dό) δ 12.80-11.80 (br m), 9.09 (s, IH, OH), 8.55 (s, IH, pteridinyl 7-CH), 8.01 (d, IH, J=7.9, amide NH), 7.97 (d, IH, J=8.1, amide NH), 7.78 (br s, IH, NH2), 7.68 (d, 2H, J=8.9, aromatic CH), 7.57 (br s, IH, NH2), 6.93 (d, IH, J=1.6, aromatic CH), 6.85-6.64 (m, 5H, aromatic CH, NH2), 6.60 (d, IH, J=8.2, aromatic CH), 4.77 (s, 2H, pteridinyl-CH2), 4.42 (m, IH, methine), 4.29 (m, IH, methine), 3.19 (s, 3H, N-CH3), 2.83 (m, 2H, ArCH2), 2.23 (t, 2H, J=7.8, glu 4-CH2), 1.90 (m, 2H, glu 3-CH2), 1.25 (s, 9H, t-Bu). Elemental Analysis: Calcd. for C33H39N9O7 2.3 H2O (MW 715.16): C, 55.42; H, 6.14; N, 17.63. Found: C, 55.58; H, 5.97; N, 17.47. Mass Spectrum: (Ion Spray) 674 (M+H) + . UN Spectrum: (pH 7 Buffer) λ ma χ (ε) 259.4 (23700), 307.4 (23500), 372.5 (7330). λmin (ε) 242.8 (14900), 273.0 (17000), 344.7 (5660). sh (e) 222.1 (26400), 287.2 (19600). Example 206 Sequencing of Human Carboxypeptidase Al cDNA: A Hind m - Sal I cDNA fragment encoding the rat preprocarboxypeptidase Al (CPAl) (Gardell et al, Nature, 317:551-555, 1985) was isolated from pMP36 provided by Dr. M. Phillips (UCSF). The 1.2 kb Hind m - Sal I rat CPAl cDNA was radiolabeled with [32p]dCTP and Prime-it kit (Stratagene) and then used to screen a lambda gtl 1 human pancreas cDNA library (Clontech) according to the method of Grunstein and Hogness (Proc. Nat. Acad. Sci 72: 5016-5020, 1975). Purified isolated plaques that hybridized to the rat CPAl cDNA probe were obtained after three rounds of screening. Lambda DNA from purified plaques was prepared from overnight growth liquid cultures using Qiagen columns and according to the manufacturer's protocol (Qiagen). Human CPAl cDNA inserts were liberated from lambda DNA by digestion with EcoRI and purification of the cDNA inserts by low-melting agarose gel electrophoresis. The EcoRI cDNA inserts were cloned into the EcoRI site of pGEM 7z(+) (Promega) and denoted as pHCPA plasmids. Following overnight growth in liquid culture, plasmid pHCPA DNA was prepared using Qiagen columns. DNA sequencing of isolated HCPA1 lambda plaques and of plasmids containing HCPA1 cDNA was performed with [35s]dATP and the finol sequencing system (Promega). Oligonucleotide primers that flanked the cloning site of lambda gtl 1 or the multiple cloning sites in pGEM 7(+) and the TA vector (Invitrogen) were used initially to determine cDNA sequence. Oligonucleotide primers that corresponded to regions of the determined sequence were synthesized which permitted the entire cDNA sequence of both strands of HCPA1 to be determined. (Seq ID 1) Example 207 Cloning of Human Carboxypeptidase A2 Human carboxypeptidase A2 (CPA2) was cloned from a human pancreas cDNA library constructed in lambda gtl l (Clontech). The probe used was an 1198 base fragment of rat CPA2, This probe was amplified from QUICK-Clone rat pancreas cDNA (Clontech) using PCR. The upstream primer corresponded to the sequence between positions 12-33 in the rat cDNA coding sequence (5'-CCTGTTATTGGCTGCCCTACTT-3'), while the downstream primer was the complement to the sequence between positions 1888-1209 (5'-AAGCCAGGTCTCTTCTGCTGTC-3')(Gardell, S.J. J.Biol. Chem 263 17828 (1988)). The standard PCR conditions were a 94°C preincubation followed by 30 cycles of one minute at 94°C, two mins at 56°C, and three mins at 72°C. The 1198 bp PCR product was extracted with 100 ul chloroform and the upper aqueous phase containing the DNA was passed through a CHROMA SPIN- 100 column (Clontech) to remove the primers. The sequence of the rat CPA2 PCR product was confirmed with DNA sequencing using the PCR primers as sequencing primers. Sequencing of the DNA was done with [ 2 P] dATP using the finol sequencing kit (Promega). The rat CPA2 DNA was radiolabelled with [ 32 P] dCTP and the Prime-it II Random Primer Labelling kit (Stratagene) and then used to screen the human pancreas cDNA library. The library was plated using E. coli strain Y1090r on 150 mm plates (approximately 10,000 plaques/plate) and duplicate nitrocellulose lifts made. The filters were blocked in hybridization solution (50% formamide, 5X SSPE, 50X Denhardt's solution, 0.1% SDS, and 100 ug ml sonicated salmon sperm DNA) for 3 hours at 42°C. Labelled rat CPA2 PCR product was added and hybridized to the filters overnight at 45 °C. The filters were washed for 1 hour at 60°C in 0.1X SSC, 0.1% SDS and exposed to autoradiography film while moist. One duplicate plaque from each plate was picked and subjected to another round of screening as described above. Positive plaques after the second round of screening were picked and the size of the lambda gtl 1 insert DNA was determined using PCR. Lambda gtl l forward and reverse sequencing primers (Clontech) that flanked the EcoRI cloning site of lambda gtl 1 were used as the upstream and downstream PCR primers, respectively. Well-isolated plaques were picked, with a portion suspended in 10 ul water, and heated at 95 °C for 5 min, and the remainder being used to make a phage stock for future screening. The 10 ul containing the plaque DNA was used directly as the template in PCR using the conditions described above. Amplified insert DNA was visualized on agarose gels and the two plaques giving the largest insert DNA were screened a third time. Lambda DNA from the two plaques was isolated and sequenced in both directions using the finol kit (Promega) and the lambda gtll forward and reverse primers (Clontech). One clone contained regions highly homologous to the 5' and 3' regions of rat CPA2 and was chosen for further study. The insert cDNA was liberated from the lambda DNA by digestion with EcoRI, gel purified, and cloned in the EcoRI site of the pGEM-7Zf(+) vector (Promega). Sequencing was carried out using [ ^] dATP and the finol kit (Promega) as described above. Oligonucleotide primers that corresponded to regions of the determined sequence were synthesized which permitted the entire cDNA sequence of hCPA2 to be determined. The cDNA was found to contain an open reading frame coding for a protein of 417 amino acids. This protein shares 87% identity with rat CPA2 at the amino acid level and 85% identity at the cDNA level. This protein shares 63% identity with human CPAl at both the amino acid and nucleotide levels. Thus the protein was classified as human carboxypeptidase A2. Table 4 shows a comparison between the amino acid sequences of human CPAl and CPA2. The amino acid numbering scheme used throughout this document is that of CPAl. All mutations described for CPA 1 and CPA2 refer to this numbering scheme. Example 208 Expression of HCPA1 in Yeast: The size of cDNA inserts within isolated HCPA1 lambda plaques was determined using oligonucleotide primers that flanked the EcoRI cloning site of lambda gtl l and polymerase chain reaction (PCR). Single isolated, plaques were picked, suspended in 200μl water and heated at 95°C for 5 minutes. Ten μl of each suspension was used for PCR amplification according to manufacturer's instructions (Perkin Elmer/Cetus). The standard PCR reaction conditions consisted of 1 minute at 95°C, .5 minute at 50°C, and 3 minutes at 72°C. The reaction products were visualized following agarose gel electrophoresis. CPA-11 5'-gCT gAA gCT TCg gAg gAC TTT-3' CPA-12 5'-TCT TgA CCg CCT ggA TgC Tg-3' The expression of HCPA1 in S. cerevisae was based on the strategy employed by Gardell et al (Nature, 317; 551-555, 1985). Two oligonucleotide primers, CPA 11 and 12, were used to amplify a 200 bp fragment from pHCPA by PCR as described above. The amplified fragment contained a new 5' Hind HI site created by CPA 11 that would allow ligation in frame with the alpha factor leader of pMP36 as described by Phillips et al (J. Biol. Chem. 265; 20692-20698, 1990). The PCR fragment was ligated into the TA vector (Invitrogen) and the entire DNA sequence of the fragment determined as described earlier. A colony that contained the desired 5' alteration was selected for further use, TAHCPAexp. Plasmid pHCPA was restricted with Aat II and EcoRI to liberate a 1.1 kb HCPA1 fragment. This fragment was purified following low melting point agarose gel electrophoresis and ligated into TAHCPAexp that had been restricted with Aatll amd EcoRI to yield the plasmid TAHCPA. TAHCPA was digested with EcoRI, treated with T4 DNA polymerase, and Sal I linkers were added and ligated to the 3' end of HCPA1 as described by Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989). TAHCPA that now contained 5* Hind m and 3' Sal I sites was restricted with Hind HI and Sal I to liberate a 1.2 kb HCPA1 cDNA fragment. This fragment was ligated together with the large Hind m fragment and the small Hind Hi-Sail fragment of ρMP36 to produce pMP36 HCPA1. Yeast expression of the cloned human enzyme is based on the method of Phillips, M. et al, J. Biol. Chem., 265, 20692 (1990). Plasmid pMP36 containing HCPA1 was restricted sequentially with BAMH1, SAL1 and SSP1 with intervening purifications by either phenol extractions or use of Promega Magic Mini Columns with manufacturer supplied procedures. Alternatively, the One-Phor-All buffer system (Pharmacia) can be used in a concurrent restriction by all three enzymes. Following SSP1 restriction, the mixture ws separated on a 1% low melting gel giving 5 distinct bands. The band of interest at about 2.8 kb (the second from top) was excised from the gel and purified using a Schleicher and Schuell Elutip-D column with the supplied procedures. The BAM/SAIJSSP fragment was then ligated into pBS24.1 shuttle vector (produced from pBS24.1-AGC3 kindly provided by Dr M Phillips, UCSF with BAMH1/SAL1 restriction) overnight at 16°C using the T4 ligase system (Gibco BRL). The ligated vector was precipitated with ammonium acetate and ethanol at -80°C for 4 hrs and resuspended in lOμl TE. 4μl of the vector was electroporated into 40μl of DH5alpha E. coli using a Bio Rad Gene Pulser at 2000 volts with a time constant around 4. One ml of Miller Hinton broth was added and the sample incubated at 37°C for one hour. Three lOOμl samples of each was plated out onto ampicillin containing LB plates. The resultant colonies were replated on fresh plates. 100ml volumes of Mueller- Hinton/ampicillin broth were innoculated with colonies and incubated overnight at 37° C. The plasmid was extracted using the Qiagen Maxi extraction procedure. Approximately 500 to 2000ng of the PBS24.1 plasmid DNA extracted above was electroporated into 40 μl of competent DLM101 alpha yeast cells. One ml of sorbitol was added immediately after electroporation to rescue the cells. 100 μl samples were plated out on dishes of YNB (yeast nitrogen broth) lacking uracil which were incubated at 30°C. Positive clones were picked. Transformants were grown in minimal synthetic medium (Wickersham, L.J., U.S. Dept. of Agric Tech. Bull. No. 1029 (1951), lacking leucine, for 48 hrs. This culture was then used to seed a 350 litre fermenter. Example 209 Purification of HCPAl or its mutants from veast: A 350 1 culture of proCPA-producing S. cerevisiae (T268G/p2619) was grown in a 500 1 New Brunswick fermentor for 65 h at 30°C with 175 1pm air flow, 5 psig head pressure and 150 rpm agitation. The medium contained 1% yeast extract (Difco), 2% tryptone (Difco), 1% glucose, 20 mM KPO4, pH 7.5, 50μg/ml ampicillin and 0.007% (v/v) Mazu DF 60P antifoaming agent (Mazei' Chemicals, Inc.). The final OD000 as 14. After cooling to 15-20°C the cells were removed from the medium by cross- flowfiltration on a Sartorius Sartocon II system equipped with 7 0.6m2 polyolefin filter units. The protein in the medium was concentrated with 10K MWCO cross-flow filters and the fluid exchanged by diafiltration with 20mM Tris-Cl. pH 8, 0.1 mM Zn-acetate (buffer A). All subsequent buffers contained 0.1 mM Zn-acetate. Ammonium sulfate (AS) was added to 0.5M to the 10K concentrate and it was loaded onto a 250 ml radial flow Phenyl-sepharose column equilibrated in buffer A with 0.5 M AS. The proCPA was eluted with a 1.5 1 .5-0 M AS gradient followed by a buffer A wash. The Phenyl- Sepharose procedure was repeated with the breakthrough material to recover unbound proCPA from the first run. Fractions containing proCPA were pooled and concentrated and dialyzed on a Sartorius 10K Easy Flow filter (0.2m 2 ) into buffer A. This was loaded onto a Hi Load 26/10 Q-Sepharose High Performance (Pharmacia) column (53ml) and eluted with a 1 litre 0-0.5M NaCl gradient in buffer A. The proCPA eluted between 120-220 mM NaCl with the peak tube at ~ 150 mM. Fractions containing proCPA were pooled and the proCPA was cleaved to mature CPA with trypsin (2μg/ml) at 37° C for 1 h. PMSF was added (0.5 mM) and the pool was dialyzed in buffer A. The Q-Sepharose HP step was repeated and the activated CPA eluted between 80-140 mM with the peak tube at 120 mM. Previous IEF experiments have shown that proCPA has a pl=4.75 while the mature CPA has a pl=6.2. The CPA fractions were pooled and concentrated with 10K Centripreps (Amicon) to 15 ml and this was passed through a FfiLoad 26/60 Superdex 75 (Pharmacia) column in 3 runs (5 ml each). The elution buffer was buffer A with 150mM NaCl. The CPA fractions were pooled, concentrated with 10K Centripreps and exchanged with Sigma PBS containing 0.1 mM Zn-acetate. The final volume (15ml) was passed through a 0.2μ syringe filter and stored at 4°C. The protein was 24.5 mg/ml by the Lowry assay using BSA standards. The specific activity for a typical preparation of the mutant with Gly at 268 was 163 u/mg. Total protein = 368 mg. Example 210 Expression of HCPA2 in Yeast The expression of HCPA2 in S. cerevisiae was employed the same strategy described for HCPA1 (Example 208) with the following exceptions: 1) the PCR primers used to introduce the new 5' Hind m site were : YEAST 1: 5'-gCAgAAgCTTCAgAAACgTTTgTgggAgATCAAgTTCTT gAgATTgTACC-3' YEAST2: 5'-CTCTTTgTCCAACAggACCTg-3' 2) a Hinc II site rather than Aat II was used to subclone the mutagenic PCR fragment to the 5' end of the HCPA2 cDNA, and 3) the cloned PCR product was cut from the TA vector and subcloned into the pGEM-7Zf(+) vector containing the HCPA2 cDNA. PCR was carried out with the YEAST 1 and 2 primers as described for HCPA1. The mutagenic PCR product was cloned into the TA vector and the sequence confirmed. This fragment was cut from the TA vector using Hind III and Hinc II and cloned into the corresponding sites of pHCPA2 and the resulting construct designated pHCPA2-Y. The unique 3' Eco RI site of pHCPA2-Y was changed to a Sal I site using linkers as described above with the resulting construct designated pHCPA2-YSal I. The 1.2 kb Hind m/Sal I HCPA2 fragment derived from pHCPA2-YSal I was excised from the vector and ligated together with the large Hind Hi/Sal I fragment of pMP36 (Phillips, M. et al, J. Biol. Chem., 265, 20692-20698 (1990)) to produce pMP36HCPA2. Yeast expression of clone HCPA2 was as described above for HCPA1 (Example 144). Human CPA2 has two internal Bam HI sites not present in human CPAl so partial digestion was needed for the final subcloning step. pMP36HCPA2 was digested to completion with Sal I and partially digested with Bam HI (as described above) to yield the 2.7 kb fragment containing the alcohol dehydrogenase/GADPH promotor and regulatory region (A/G promotor) attached to human CPA2 fused in frame to the yeast α factor leader. This fragment was ligated into the Sal I/Hind m site of pBS24.1 forming pBSHCPA2. The construct was amplified in DH5α and used to electroporate yeast as described above (Example 208). Example 211 Purification of HCPA2 or its Mutants from Yeast An overnight culture (grown in Leu" medium) of S. cerevisiae expressing proCPA2 was used to start 2 1 of culture in 1% yeast extract (Difco), 2% tryptone (Difco), 1% glucose, 20 mM KPO4 , pH 7.5, and 50 μg/ml ampicillin. The culture was grown for 48 hrs at 30°C and the supernatant cleared by centrifugation at 6000 X g for 10 min at 4°C. The protein in the supernatant was concentrated on an Amicon PM-10 Ultrafiltration membrane to 114 ml. The concentrated supernatant was made to 20 mM Tris-HCl (pH 8.0), 0.1 mM Zn-acetate and 0.5 M ammonium sulfate and loaded onto a 1.8 X 20 cm Phenyl-Sepharose column (Pharmacia) equilibrated in buffer A with 0.5 M ammonium sulfate. The protein was eluted with a 120 ml linear gradient (1 ml/min) from buffer B (20 mM Tris-HCl pH 7.5, O.lmM Zn-acetate) with 0.5 M ammonium sulfate to buffer B with 20 % (v/v) ethanol. Fractions containing carboxypeptidase activity were pooled and dialyzed against buffer B. The dialyzed pool was loaded onto an HR 5/5 (1 ml) Mono Q column (Pharmacia) and eluted with a 30 ml linear gradient (lml/min) from 0- 0.5 M NaCl in buffer B. Fractions containing carboxypeptidase activity were pooled and dialyzed against buffer B. The pooled proCPA2 was activated with trypsin (10 μg/ml) at 37°C for 1 hr and the trypsin inactivated with PMSF added to 0.5 mM. The mature CPA2 was dialyzed against buffer B and again loaded onto the 1 ml Mono Q column (Pharmacia) and eluted as above. Fraαions containing carboxypeptidase activity were pooled, dialyzed against buffer B, and concentrated 10 fold in an Amicon Ultrafiltration cell using a PM-10 membrane. The concentrated mature CPA2 was loaded onto an HR 10/30 Superose 12 column (25 ml) and eluted isocratically (0.4 ml/min.) in phosphate buffered saline (10 mM Na2HPO4, 0.15 M NaCl, pH 7.2). For wild type HCPA2 the protein was 0.65 mg ml (4.13 mg total) and for the 268 Gly mutation the concentration was 0.3 mg/ml (4.47 mg total) as determined with the Micro BCA Assay (Pierce Chemical Co.) using BSA standards. Example 212 Expression of Mutant HCPA2 The A250G and T268G mutations of HCPA2 were constructed as described above (Example 149) for mutant forms of HCPA1 using the T7-Gen In Vitro Mutagenesis Kit (United States Biochemical, No. 74500). The mutagenic oligonucleotide primers (Oligos Etc.) to mutate the A250 (gCC) and T268 (ACC) to Gly (ggC ) are shown below: A250G 5' - TCC TCC ACT gCC TTg gTA gAT - 3' Gly = ggC T268G 5' - CAg TTC AAA gCC AAA TgA gTA - 3' Gly = ggC Mutagenic codons are underlined. Using these oligonucleotide primers the following mutant forms of HCPA2 were produced: 1. A250G 2. T268G Each of the above mutagenized HCPA2 cassettes was sequenced to verify that only the desired DNA mutations were produced. The mutagenized cassettes were subcloned into pMP36HCPA2 that could further be processed for expression in S. cerevisiae using methods described in Example 152. Example 213 Svnthesis of a Chemical conjugate between Campath-1-H or ING-1 and Human carboxypeptidase A mutants 2mg of mutant carboxypeptidase A was combined with 0.15 mg of Sulfo-SMCC (Pierce Chemical Co) in 475 μl of Dulbecco's Phosphate Buffered Saline (PBS). The resulting solution was stirred for 45 minutes at 25°C. The modified enzyme was purified through a 1 x 13 cm G-25 medium column equilibrated with PBS. Purified product can be stored at 4°C for 24 hrs. Maleimide content was determined by combining 0.5 ml of 6.3 μM modified enzyme with 6 μl of ImM mercaptoethanolamine. After 30 minutes, 20 μl of 4 mg/ml Ellman's reagent was added, and after another 20 minutes absorbance at 412 nm was determined. Based upon a molar absoφtivity of 13.6 mM -1 , the purified product was found to contain 1 maleimide per enzyme molecule. The modification had no effect upon enzyme activity. The antibody was modified with 2-iminothiolane (Pierce Chemical Co). 0.35 mg of antibody solution was combined with 0.055 mg of 2-iminothiolane in 0.1 M triethanolamine-HCl, 2mM EDTA, pH 8.0 under anaerobic conditions, reacted with stirring for 1 hr and 45 mins. The modified antibody was purified through a 1 x 13 cm G-25 medium column equilibrated with 0.02 M sodium acetate, 0.1 M NaCl, pH 5.8, bubbled with and maintained under a He atmosphere. The purified modified antibody solution was collected directly into the solution of modified carboxypeptidase A. The resulting solution was adjusted to pH 7.4 with NaOH, made anaerobic, and allowed to react, with stirring, at 4°C for 18 hours. (Optionally free maleimide groups may be removed by reacting the solution with 0.3 mM mercaptoethanolamine at room temperature for one hour). The solution concentrated to 1 ml. The resulting concentrated conjugate was purified from aggregates, unreacted enzyme and small molecules by chromatography on Superose 12 HR 10/30. The enzyme specific activity of the conjugate was found to be appropriate for a one to one conjugate with antibody. Antibody (Campath-IH® or ING-1) % immunnoreactivity and antigen binding affinity of the conjugate were both found to be high. Example 214 The enzyme was modified by the protocol described for Example 4 above, while the antibody was modified with dithiothreitol. 0.7 mg of antibody solution was combined with 0.035 μmol of dithiothreitol in 350 μl of 0.1 M triethanolamine-HCl, 2 mM EDTA, pH 8.0 under anaerobic conditions and reacted with stirring for 1 hr. The modified antibody was purified through a 1 x 13 cm G-25 medium column equilibrated with 0.02 M sodium acetate, 0.1 M NaCl, pH 5.8, bubbled with and maintained under a He atmosphere. The purified antibody solution was collected directly into the solution of modified carboxypeptidase A. The resulting solution was adjusted to pH 7.4 with NaOH, made anaerobic, and allowed to react, with stirring, at 24°C for 30 min and at 4° C for 18 hrs. Then the solution was concentrated to 1ml. The resulting concentrated conjugate was purified from aggregates, unreacted enzyme and small molecules by chromatography on Superose 12 HR 10/30. The enzyme specific activity of the conjugate was found to be appropriate for a one to one conjugate with antibody. Antibody % immunoreactivity and antigen binding affinity were both found to be high. Example 215 The enzyme was modified by the protocol described for Example 4 above, while the antibody was modified with SATA (Pierce Chemical Co). 0.225 mg of antibody solution was combined with 0.003 mg of SATA in a total volume of 150 μl of Dulbecco's Phosphate Buffered Saline (PBS) and allowed to react with stirring for 45 mins. The modified antibody was purified through a 1 x 13 cm G-25 medium column equilibrated with PBS. The purified modified antibody solution was mixed with the modified enzyme and made anaerobic, 300 μl of anaerobic 0.5 M NH2OH was added and the solution allowed to react, with stirring, at 25°C for 2 hrs followed by 4°C for 18 hrs. Free maleimide groups were removed by reacting the solution with 0.3 mM mercaptoethanolamine at room temperature for one hour, and the solution was then concentrated to 1 ml. The resulting concentrated conjugate was purified from aggregates, unreacted enzyme and small molecules by chromatography on Superose 12 HR 10/30. The enzyme specific activity of the conjugate was found to be appropriate for a one to one conjugate with antibody. Antibody antigen binding affinity was found to be high. Example 216 Expression of Mutant HCPA1 pMP36 containing wild type (WT) proHCPAl cDNA (as a fusion with yeast alpha-factor leader described above) was restricted with Nco I and Sal I to liberate a 481 bp cDNA fragment. This fragment begins at nucleotide 893, proceeds to the very 3' end of HCPA1 cDNA and encodes amino acids 186-309 of mature HCPA1. The HCPA1 Nco I-Sal I fragment was ligated into the Nco I and Sal I cloning sites of pGEM5zf(-) (Promega) to generate pHCPAINS (Figure 3). pHCPAINS was then restricted with Sph I and Sal I to liberate the HCPA1 Nco I-Sal I fragment and additional (9 bp) pGEM5zf(-) sequence.. This Sph I-Sal I fragment was cloned into M13n_.pl 9 (BRL) using its Sph I and Sal I cloning sites to generate M13mpl9HCPAl (Figure 4). Single stranded M13mpl9HCPAl DNA was used as template for olignucleotide-directed mutagenesis using the T7-GEN In Vitro Mutagenesis Kit (United States Biochemical, No. 74500). Mutagenic oligonucleotide primers (Oligos Etc) listed below were used to mutate residues 1255 (ATT) and T268 (ACC) either separately or in tandem. 1. I255A 5'- ggT CCA gTC AgC AgT gCT TCC -3' Ala = gCT 2. T268A 5'- gAg CTC gAA ggC gAA ggA gTA -3' Ala = gCC 3. T268G 5'- gAg CTC gAA gCC gAA ggA gTA -3' Gly = ggC Mutagenic codons are underlined. Using these oligonucleotide primers the following mutant forms of HCPA1 were produced: 1. I255A 2. T268A 3. T268G 4. I255A/T268A Each of the above mutagenized HCPAl cassettes was sequenced to verify that only the desired DNA mutations were produced. pMP36 HCPAl w.t. was restricted with Ncol to liberate a 1.2 kb fragment that was cloned into the Nco I site of M13mpl9HCPAl mutants. After the correct orientation of the Nco I fragment within M13mpl9 proHCPAl mutants (Figure 5) was verified the DNAs were restricted with Hind m and Sal I which liberated a 1.2 kb cDNA fragment encoding the entire proHCPAl mutant enzyme. This fragment was then ligated into the Hind m and Sal I sites of pMP36 yielding pMPHCPAl mutants (Figure 6) that could then be further processed for expression in f cerevisae using the methods described in Examples 2 and 3. Example 217 Expression of Mutant HCPAl Alternatively, the mutations have been conveniently generated in hCPA in the pMP36 plasmid (pMP36 HCPAl) by the unique site elimination (USE) procedure of Clontech Laboratories, Inc. using the manufacturers' reagents and protocols. In the protocol, elimination of a unique EcoRI in the pMP36 HCPAl plasmid was used for selection with the oligonucleotide XS5 (TCC CCC GGG CTG CAG GAT ATC GAT AGC TGG GCT GTG T). The oligonucleotides used to generate three specific mutations in hCPA by this protocol follow: T 268 H (ACC codon to CAC) with oligonucleotide XT2 (ATC AAG TAC TCC TCC CAG CTC CGG GAC) S 253 G (AGC codon to GGT) with oligonucleotide XT3 (TTT ATC AAG CCA GTG GAG GTA TTG ACT GGA CC) A 250 G (GCC codon to GGC) with oligonucleotide XT4 (AAG GC A ATT TAT CAA GGC AGT GGA AGC ACT ATT) The mutations were isolated using the oligonucleotides listed above and XS5 (TCC CCC GGG CTG CAG GAT ATC GAT AGC TGG GCT GTG T) which eliminates a unique EcoRI site. Plasmids containing the desired mutations were selected by digesting the wild type pMP36(hCPA) with EcoRI (uncut plasmids transform 10 to lOOx more efficiently than linearized plasmid). In addition, the XS5 selection oligonucleotide introduces an EcoRN site which can then be used to screen for mutant plasmids. In the mutant plasmids the EcoRN is unique and can therefore be used as a selection site to construct double mutants with an oligonucleotide that converts the EcoRN site back to an EcoRI site. The mutant pMP36 HCPA so generated could then be further processed for expression in S. cerevisae using the methods described in Examples 2 and 3. Example 218 Stability and biodistribution of prodrugs in vivo: Mice were dosed i.p. or i.v. bolus with 50 mg/kg of prodrug by administration of a 5 mg/ml solution in PBS, pH 6-8. Animals were sacrificed after 30 min and 2 h and the plasma, and tissues were collected. Plasma was frozen at -70°C and tissues were snap frozen in liquid nitrogen and stored at -70°C. Duplicate animals were sacrificed for each time point. Plasma was prepared for extraction by 4-fold dilution with ice cold 0.1 Ν HCl. Tissues were prepared for extraction by homogenization in 5 volumes of ice cold 0.1 Ν HCl with a Polytron equipped with a PTA 7 generator (Brinkman Instruments, Westbury, ΝY). Drugs were subsequently extracted by adding 500 μl of -20°C acetonitrile to 200 μl of the 0. IN HCl homogenate. After a 5 min incubation on ice, samples were centrifuged at 12,400 x g for 15 min. Supernatants were collected and diluted 3.57 fold with PBS to reduce the final acetonitrile concentration to 20%. Diluted supernatants were then analyzed by HPLC as described below. Drug recovery was estimated by spiking control tissue homogenates with either drug or prodrug prior to the acetonitrile extraction step. After correction for recovery of these standards, the levels of drug and prodrug in tissues and the stability of the prodrug in these tissues were calculated using HPLC. Samples were analyzed on a 4 μm C18 Νova-Pak 3.9 x 300 mm steel HPLC column equipped with a C18 guard column with UN detection at a flow rate of 1 ml min. Samples were injected onto the HPLC column equilibrated with 2% acetic acid, pH 3.0, containing either 5% or 20% acetonitrile depending upon the compound being analyzed. Upon sample injection a 25 or 35 min linear gradient was initiated raising the acetonitrile concentration to 50% acetonitrile. Three compounds (ASP-MTX, o-cyclopentyl-PHE- MTX and m-cyclopentyl-PHE-MTX) were analyzed on a 10 μm C18 uBondapak column (3.9 x 300 mm) equilibrated with 16% acetonitrile in 5 mM tetrabutylammonium hydrogen sulfate, 10 mM NH4H2PO4. The column was then eluted with a 30 min linear gradient of up to 50% acetonitrile. Results indicated in Table 1. The results show that a compound that is a good substrate for wild type CPA, such as compound # 10, is not stable in vivo. This type of compound is not useful for ADEPT. However, compounds which are good substrates for a mutant enzyme but not for the wild type enzyme, such as compounds 8, 9, 11, 12, 13 and 14 and others, are stable in vivo and are as such useful in ADEPT. The data shown in Table 1 indicate the concentration of prodrug detected over time in minutes as either μM in the case of plasma or nmol g in the case of tissues. The figures in brackets indicate the stability of the prodrug expressed as the percentage of material (sum of drug and prodrug) observed as intact prodrug. Except as indicated all experiments were carried out in CD-I nude mice. D Table 1 D Table 1 (Continued! Prodrugs 1 to 14 shown in Table 1 are as follows: 1. N-(N-(4((( 1 ,2-dihydro-3-methyl- 1 -oxobenzo(F)quinazolin-9-yl)methyl)amino)-2- fluorobenzoyl)-L-glutam- 1 -yl)-L-glutamic acid 2. N-((S)-4-carboxy-2-(5-((( 1 ,2-dihydro-3-methyl- 1 -oxoxbenzo(F)quinazolin-9- yl)methyl)amino)- 1 -oxo-2-isoindolinyl)butanoyl)-L-phenylalanine 3. N-(N-(2-fluoro-4-((( 1 ,2-dihydro-3-methyl- 1 -oxobenzo(F)quinazolin-9- yl)methyl)amino)benzoyl)-L-glutam- 1 -yl)-3-( 1 -naphthyl)-L-alanine 4. N-(N-(4(((l,2-dihydro-3-methyl-l-oxobenzo(F)quinazolin-9-yl)methyl)amino)-2- fluorobenzoyl)-L-glutam- 1 -yl)-2-carboxy-L-phenylalanine 5. N-(N-(4-((( 1 ,2-dihydro-3 -methyl- 1 -oxobenzo(F)quinazolin-9-yl)methyl) amino)-2-fluorobenzoyl)-L-glutam- 1 -yl-2-iodo-L-phenylalanine 6. N-(4*^(2,4-di_unino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-L- aspartic acid 7. N-(4-((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-2- carboxy-L-phenylalanine 8. N-(4-((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-2- cyclopentyl-L-phenylalanine 9. N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3- cyclopentyl-L-phenylalanine 10. N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl- L-pheylalanine. 11. N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl- 3-cyclobutyl-L-phenylalanine. 12. N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3- tert-butyl-L-phenylalanine. 13. N-(4-(((2,4-di__mino-6-pteridinyl)methyl)methylanιino)benzoyl)-L-glutam-l-yl-3- cyclopentyl-L-tyrosine. 14. N*^4*-((3-(2,4-diainino-l,6-dihydro-6-oxo-5-pyrimidinyl)propyl)amino)benzoyl)- L-glutam- 1 -yl-3 -cyclopentyl-L-phenylalanine. Example 219 Molecular modelling of human CPAl: The Brookhaven Protein Database (Bernstein F.C. et al, J. Mol. Biol. 112, 535-542 [1977]) was searched and bovine CPA selected as that likely to have the greatest structural similarity to human CPA (HCPA) based on base sequence homology. The COMPOSER algorithm which was used to develop the HCPA model is part of the commercially available SYBYL molecular modelling package sold by TRIPOS Associates (SYBYL Molecular Modeling Software, Version 5.3, November 1989, TRIPOS Associates Inc., St Louis, MO 63144). The COMPOSER algorithm was used to fit the amino acid residues of the human CPA sequence into the 3D coordinates of the crystal structure of bovine CPA to determine a best fit and consequently a projected 3D structure for HCPA. Example 220 Cell Culture Targetting and Therapy Wein-133 B-cell lymphoma cells were incubated with a conjugate of wild-type HCPAl and Campath for 30 min. Cells were then washed to remove unbound conjugate and returned to cell culture at 500,000 cells mH. Cells were cultured in the presence of varying concentrations of the prodrug MTX-Phe and levels of inhibition of growth analysed after 3 days. Results are illustrated in Fig 1. Similar results were obtained when a conjugate of mutant 268 Gly with Campath was substituted for the wild type conjugate. Example 221 Prodrug toxicitv The stable prodrug of the present invention should in addition to being stable in vivo also be less toxic in the host than the parent drug in order to permit larger dosing levels of prodrug and in turn generation of local high concentrations of active agent. We have found that the maximum tolerated dose for methotrexate in the mouse (female, strain CD-I nu/nu) is 4 mg/kg IN once daily for 5 days. Doses of Ν-(4-(((2,4-diamino-6- pteridinyl) methyl) methylamino) benzoyl)-L-glutam-l-yl-2-cyclopentyl-L-phenylalanine up to 20 mg/kg IN once daily for 5 days were tolerated, and doses of up to 40mg/kg of N-(4-(((2,4* iiamino-6-pteridinyl) methyl) methylamino) benzoyl)-L-glutam-l-yl-3- cyclopentyl-L-phenylalanine or N-(4-(((2,4-diamino-6- pteridinyl) methyl) methylamino) benzoyl)-L-glutam-l-yl-3-tert-butyl-L-phenylalanine IN once daily for 5 days produced no toxicity; therefore the maximally tolerated doses of the latter two compounds are greater than 40mg/kg. In Swiss nu nu mice, the maximum tolerated dose of methotrexate is 2.5 mg/kg IN daily for 5 days. In these mice, Ν-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3-cyclopentyl-L-tyrosine was tolerated at doses to 50 mg/kg IN daily for 5 days while Ν-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3 -cyclobutyl-L-phenylalanine showed some toxicity at doses of 30 mg/kg IN daily for 5 days. Thus, in Swiss nu/nu mice, with this daily x 5 schedule, the MTD for Ν-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3-cyclopentyl-L-tyrosine and N- (4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3- cyclobutyl-L-phenylalanine would be > 50 mg/kg and < 30 mg/kg, respectively. Thus the prodrugs of the present invention are indeed much better tolerated than the corresponding active agent. Example 222 Activity of a mutant human carboxypeptidase Al with a stable prodrug As indicated herein, the in vivo stable prodrug of the present invention should be a better substrate for a mutant enzyme than the corresponding wild type enzyme in order for the stable prodrug to be activated selectively at the tumour. Table 2 shows the enzyme kinetics for several prodrugs of methotrexate with wild type human carboxypeptidase Al and mutants with thr 268 mutated to either Ala or Gly. The most relevant kinetic constant in the table is the kcat/Km, which provides an estimate of the second order rate constant for the enzyme catalyzed conversion of prodrug to methotrexate. Thus, the larger the kcat/Km, the more efficient the enzyme with a particular substrate. It can be seen that the kcat/Km for human CPAl with one of the best substrates for this wild type enzyme N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl- L-phenylalanine the prodrug of MTX, is 0.44/uM/see The kcat/Km values for N-(4- (((2,4-diamino-6-pteridinyl) methyl) methylamino) benzoyl)-L-glutam-l-yl-2- cyclopentyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl) methyl) methylamino) benzoyl)-L-glutam- 1 -yl-3-cyclopentyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl) methyl) methylamino) benzoyl)-L-glutam-l-yl-3-tertbutyl-L-phenylalanine, N-(4-(((2,4- diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3-cyclobutyl-L- phenylalanine, and N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L- glutam-l-yl-3-cyclopentyl-L-tyrosine for the human CPAl are much lower; indeed the human CPA catalyzed reactions with the latter four prodrugs were not measureable. However, the kcat/Km for these five stable prodrugs with 268Gly are 0.157, 0.092, 0.38, 1.8 and 0.18/uM/sec, respectively. Thus, the single mutation at 268 from thr to gly converted very poor substrates in the wild type enzyme to excellent substrates in the mutant enzyme. Indeed in the case of N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glut am- 1 -yl-3 -tertbutyl-L-phenylalanine the activity of 268Gly with the prodrug is not different than the human CPAl with N-(4- (((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-L- phenylalanine, one of its best substrates. Further, in the case of N-(4-(((2,4-diamino- 6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3 -cyclobutyl-L-phenylalanine the activity of 268Gly is 4 times better than CPAl with N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-L-phenylalanine. Thus, the compounds are much better substrates for the mutant enzyme than the wild type enzyme which permits very specific tumor targeted activation of the nontoxic, stable prodrug to the active agent. Table 2 kinetic Human Prodrug constant CPAl 268ala 268gly N-(4-(((2,4-diamino-6-pteridinyl)methyl) Km (uM) 4.3 0.9 0.3 methylamino)benzoyl)-L-glutam- 1 -yl- kcat (1/sec) 1.9 2.0 2.2 L-phenylalanine kcat/Km 0.44 2.25 7.35 N*^4-(((2,4-diamino-6-pteridinyl)methyl) Km (uM) 61 56 15 methylamino)benzoyl)-L-glutam- 1 -yl-2- kcat (1/sec) 0.012 0.088 2.35 cyclopentyl-L-phenylalanine kcat/Km 0.0002 0.0016 0.157 N-(4-(((2,4-diamino-6-pteridinyl)methyl) Km (uM) ND 31 15 methylamino)benzoyl)-L-glutam- 1 -yl-3 - kcat (1/sec) ND 0.001 1.4 cyclopentyl-L-phenylalanine kcat/Km ND 0.00003 0.092 N-(4-(((2,4-diamino-6-pteridinyl)methyl) Km (uM) ND ND 2.1 methylamino)benzoyl)-L-glutam- 1 -yl-3- kcat (1/sec) ND ND 0.81 tertbutyl-L-phenylalanine kcat/Km ND ND 0.38 N-(4-(((2,4-diamino-6-pteridinyl)methyl) Km uM) ND 160 1.8 methylamino)benzoyl)-L-glutam- 1 -yl-3- kcat (1/sec) ND 0.12 3.0 cyclobutyl-L-phenylalanine kcat/Km ND 0.00075 1.8 N-(4-(((2,4-diamino-6-pteridinyl)methyl) Km (uM) ND 50 18 methylamino)benzoyl)-L-glutam- 1 -yl-3 - kcat (1/sec) ND 0.0006 3.0 cyclopentyl-L-tyrosine kcat/Km ND 0.00001 0.18 ND = not determined; rate to slow to assay, kcat < 0.0005/sec Kinetics were determined by a modification of the coupled assay of Kuefher, et al (1989) Biochemistry 28 2288-2297 in which the product MTX is converted by excess carboxypeptidase G to the pteroate of MTX. In this assay the reaction is followed spectrophotometrically at 315 nm; at this wavelength the change in molar absorbtivity for the coupled reaction was determined to be 9.57 mM-lcm"! . Assays were performed at 25°C in 25 mM tris-HCl, 100 mM NaCl, pH 7.4. Prodrugs were preincubated at 25 °C with buffer and 17 milliunits of Sigma #C-9658 carboxypeptidase G in 1 ml total volume. Assay was initiated with carboxypeptidase A or the appropriate mutant. Initial velocities were obtained, and standard Michaelis-Menten calculations were used to determine kinetic constants (in Biochemistry. Lehninger, A.L. 1970, Worth Publishers, Inc, NY). TABLE 3 Antibodies and Antigens Antigen Antibody Current uses Tumor associated Antigens Pan Adenocarcinoma ING-1 Imaging and therapy of (Wellcome) various carcinomas. Cytokeratins cl74 Imaging and therapy of (Biomira) Squamous cell carcinomas. Cytokeratins 16.88 Imaging and therapy of (Organon Teknika) Squamous cell carcinomas. Carcinoembryonic Antigen NR-CO-02 Imaging and therapy of (NeoRex) colon/gastrointestinal tumors Carcinoembryonic Antigen PR.1A3 Imaging and therapy of (ICRF) colon/gastrointestinal tumors Carcinoembryonic Antigen Immu-14 Imaging and therapy of (Immunomedics) colon/gastrointestinal tumors Carcinoembryonic Antigen Col-1 Imaging and therapy of (Dow) colon/gastrointestinal tumors Carcinoembryonic Antigen C110 Imaging and therapy of (Abbott) colon/gastrointestinal tumors Carcinoembryonic Antigen A5B7 Imaging and therapy of (Brit. J. Cancer, 1986, 54, colon/gastrointestinal 75) tumors Carcinoembryonic Antigen BW431/26 Imaging and therapy of (Br J. Cancer 1992, 65, 234) colon/gastrointestinal (Cancer Immunol tumors Immunother 1992, 34, 343) Carcinoembryonic Antigen C46.3 Imaging and therapy of (Cytogen) colon/gastrointestinal tumors Pan Adenocarcinoma MAb 12.8 Imaging and therapy of (Biochem Pharmacol 1991, various carcinomas. 42, 2062) Pan Adenocarcinoma CC49 and B72.3 Imaging and therapy of (TAG-72) (Dow) various carcinomas. Histones TNT-1 and TNT-2 All tumors with necrosis (Techniclone) Folate binding Protein MOV18 Ovarian Carcinoma (Centocor) Folate binding Protein MOV19 Ovarian Carcinoma (Int. J. Cancer 39, 297, 1987) Pan Adenocarcinoma 323/A3 Imaging and therapy of (Centocor) various carcinomas. Pan Adenocarcinoma 17.1A Imaging and therapy of (Centocor) various carcinomas. Renal cell G250 Imaging and therapy of (Centocor) Renal Cell carcinomas. 200Kd squamous cell U36 Imaging and therapy of surface antigen (Centocor) Squamous cell carcinomas. 22Kd squamous cell surface E48 Imaging and therapy of antigen (Centocor) Squamous cell carcinomas. 125Kd colon SF-25 Imaging and therapy of adenocarcinoma cell surface (Centocor) various carcinomas. glycoprotein High molecular weight Various mucin targeting Imaging and therapy of mucins antibodies (Trends various carcinomas. Biochemical Science, 1992, 11, 359) High molecular weight HMFG1 Imaging and therapy of Mucin (Cancer Research, 1992, 52, various carcinomas. 904) High molecular weight BrE-3 Imaging and therapy of Mucin (Hybridoma, 1993, 12, 15) various carcinomas. Lung squamous cell RS7-3G11 Imaging and therapy of lung carcinoma antigen (Antibody, squamous cell carcinomas. Immunoconjugates and Radiopharmaceuticals, 1991 4 703) LEY BR64 and BR96 Imaging and therapy of related tumor antigen (Bristol-Meyers Squibb) various carcinomas. HER2 protooncogene P185HER2 Imaging and therapy of product (NeoRx) various carcinomas, especially breast. human carcinoma antigen 15A82a Imaging and therapy of (Cytogen) various carcinomas. human Chorionic W14A and SB10 Imaging and therapy of gonadotropin (Int J. Cancer, 1984, 33, 429. various carcinomas. Br. J. Cancer, 1990, 6_L, 659) TABLE 4 Amino acid coding sequences for human CPAl and CPA2.* -110 -100 -90 -80 -70 HCPAl MRGLLVLSVL LGAVFGKEDF VGHQVLRISV ADEAQVQKVK ELEDLEHLQL HCPA2 ::LI:FFGA: F:HIYCL:T: ::D:::E:VP SN:E:IKNLL Q: :AQ: : : : : -60 -50 -40 -30 -20 HCPAl DF RGPAHPG SPIDVRMPFP SIQAVKIFLE SHGISYETMI EDVQSLLDEE HCPA2 :::KS:TTS: ETAH: :V: :V NV: : : :V: : : :Q::A:SI:: ::::V:::K: -10 1 11 21 31 HCPAl QEQMFAFRSR ARSTDTFNYA TYHTLEEIYD FLDLLVAENP HLVSKIQIGN HCPA2 N:E:LFN:R: E:-SGN::FG A: ::::::SQ EM:N::::H: G::::VN::S 41 51 61 71 81 HCPAl TYEGRPIYVL KFSTGGSKRP AI IDTGIHS REWVTQASGV FAKKITQDY HCPA2 SF:N::MN:: ::::::-DK: :::L:A:::A :::::: :TAL :T:N::VS:: 91 101 111 121 131 HCPAl GQDAAFTAIL DTLDIFLEIV TNPDGFAFTH STNRM RKTR SHTAGΞLCIG HCPA2 :K-PSI:S:: :A:::::LP: :::::YV:SQ TK: :::::: : :KVS::::V: 141 151 161 171 181 HCPAl VDPNRNWDAG FGLSGASSNP CSETYHGKFA NSEVEVKSIV DFVKDHGNIK HCPA2 • • • • • • • » • • • * t' P ** zi, * " *» » • • ΠQ • • • Q • • • • • • • • • • • • • T • C • • v\τ• 191 201 211 221 231 HCPAl AFISIHSYSQ LLMYPYGYKT EPVPDQDELD QLSKAAVTAL ASLYGTKFNY HCPA2 :::TL::::: :::F:::::C TKLD:F:::S EVAQK-.AQS: R: :H: : :YKV 241 251 261 271 281 HCPAl GSIIKAIYQA SGSTID TYS QGIKYSFTFE LRDTGRYGFL LPASQIIPTA HCPA2 :P:CSV:::: ::GS:::S:D Y: : : : : :A: : :::::::::: :::R:.L::: 291 301 HCPAl KETWLALLTI MEHTLNHPY HCPA2 E: : : :G:KA: : : :VRD: : : * Numbering scheme for CPAl is used. Dashes at positions 3 and 57 indicate a gap in the A2 sequence required for optimal alignment with Al . Colons indicate identity between Al and A2. Prepro-CPA begins at amino acid -110 and runs to +309. Pro-CPA begins at amino acid -94 and runs to +309. mature CPA begins at amino acid +1 and runs to +309. TABLE 5 Amino acid sequences for the 268Gly mutations of human CPAl and CPA2.* (mut.) HCPAl HCPA2 (mut. ) -60 -50 -40 -30 -20 HCPAl DF RGPAHPG SPIDVRMPFP SIQAVKIFLE SHGISYETMI EDVQSLLDEE HCPA2 :::KS.TTS: ETAH: :V: :V NV: : : :V: : : :Q::A:SI:: ::::V:::K: (mut.) -10 1 11 21 31 HCPAl QEQMFAFRSR ARSTDTFNYA TYHTLEEIYD FLDLLVAENP HLVSKIQIGN HCPA2 N:E:LFN:R: E:-SGN::FG A:::::::SQ EM:N::::H: G::::VN::S (mut. ) 41 51 61 71 81 HCPAl TYEGRPIYVL KFSTGGSKRP AIWIDTGIHS REWVTQASGV WFAKKITQDY HCPA2 SF:N::MN:: ::::::-DK: :::L:A:::A :::::: :TAL :T:N::VS:: (mut. ) 91 101 111 121 131 HCPAl GQDAAFTAIL DTLDIFLEIV TNPDGFAFTH STNRMWRKTR SHTAGSLCIG HCPA2 :K:PSI:S_: :A:::::LP: :::::YV:SQ TK: :::::: : :KVS::::V: (mut.) 141 151 161 171 181 HCPAl VDPNRNWDAG FGLSGASSNP CSETYHGKFA NSEVEVKSIV DFVKDHGNIK HCPA2 • • • • • • • • • • • • *p • _fι • • • • : :DS: : :PS: : :I:S: :KV: (mut.) 191 201 211 221 231 HCPAl AFISIHSYSQ LLMYPYGYKT EPVPDQDELD QLSKAAVTAL ASLYGTKFNY HCPA2 :::TL::::. :::F:::_:C TKLD:F:::S EVAQK.AQS: R::H:::YKV (mut.) 241 251 261 271 281 HCPAl GSIIKAIYQA SGSTIDWTYS QGIKYSFGFE LRDTGRYGFL LPASQIIPTA HCPA2 :P:CSV:::: ::GS:::S:D Y::::::G:: :::::::::: :::R::L::: (mut.) 291 301 HCPAl KETWLALLTI MEHTLNHPY HCPA2 E::::G:KA: : : :VRD: : : * Numbering scheme for CPAl is used. Dashes at positions 3 and 57 indicate a gap in the A2 sequence required for optimal alignment with Al . Colons indicate identity between Al and A2. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: The Wellcome Foundation Limited (B) STREET: Unicorn House. 160 Euston Road (C) CITY: London (D) STATE: not applicable (E) COUNTRY: England (F) POSTAL CODE (ZIP): NW12BP (A) NAME: SMITH. Gary Keith (B) STREET: 521 Mills Street (C) CITY: Raleigh .. (D) STATE: North Carolina " (E) COUNTRY: United States of America (F) POSTAL CODE (ZIP) : 27608 (A) NAME: BLUMENKOPF. Todd Andrew (B) STREET.- 201 Hunter Hill Road (C) CITY: Chapel Hill (D) STATE: North Carolina (E) COUNTRY: United States of America (F) POSTAL CODE (ZIP): 27516 (A) NAME: CORY. Michael (B) STREET: 55 Cedar Street (C) CITY: Chapel Hill (D) STATE: North Carolina (E) COUNTRY: United States of America (F) POSTAL CODE (ZIP): 27514 (ii) TITLE OF INVENTION: THERAPY (iii) NUMBER OF SEQUENCES: 22 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0. Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1257 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: (A) NAME/KEY: " CDS (B) LOCATION: 1..1257 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG CGC GGG TTG CTG GTG πG AGT GTC CTG πG GGG GCT GTC Tπ GGC 48 Met Arg Gly Leu Leu Val Leu Ser Val Leu Leu Gly Ala Val Phe Gly 1 5 10 15 AAG GAG GAC ΗT GTG GGG CAT CAG GTG CTC CGA ATC TCT GTA GCC GAT 96 Lys Glu Asp Phe Val Gly His Gin Val Leu Arg lie Ser Val Ala Asp 20 25 30 GAG GCC GAG CAC CTG 144 Glu Ala Glu His Leu CAG CTG GAC πC TGG CGG GGC CCT GCC CAC CCT GGC TCC CCC ATC GAC 192 Gin Leu Asp Phe Trp Arg Gly Pro Ala His Pro Gly Ser Pro He Asp 50 55 60 GTC CGA ATG CCC πC CCC AGC ATC CAG GCG GTC AAG ATC TTT CTG GAG 240 Val Arg Met Pro Phe Pro Ser He Gin Ala Val Lys He Phe Leu Glu 65 70 75 80 TCC CAC GGC ATC AGC TAT GAG ACC ATG ATC GAG GAC GTG CAG TCG CTG 288 Ser His Gly He Ser Tyr Glu Thr Met He Glu Asp Val Gin Ser Leu 85 90 95 CTG GAC GAG GAG CAG GAG CAG ATG πC GCC πC CGG TCC CGG GCG CGC 336 Leu Asp Glu Glu Gin Glu Gin Met Phe Ala Phe Arg Ser Arg Ala Arg 100 105 110 TCC ACC GAC ACT Tπ AAC TAC GCC ACC TAC CAC ACC CTG GAG GAG ATC 384 Ser Thr Asp Thr Phe Asn Tyr Ala Thr Tyr His Thr Leu Glu Glu He 115 120 125 TAT GAC πC CTG GAC CTG CTG GTG GCG GAG AAC CCG CAC CTT GTC AGC 432 Tyr Asp Phe Leu Asp Leu Leu Val Ala Glu Asn Pro His Leu Val Ser 130 135 140 AAG ATC CAG Aπ GGC AAC ACC TAT GAA GGG CGT CCC Aπ TAC GTG CTG 480 Lys He Gin lie Gly Asn Thr Tyr Glu Gly Arg Pro He Tyr Val Leu 145 150 155 160 AAG πC AGC ACG GGG GGC AGT AAG CGT CCA GCC ATC TGG ATC GAC ACG 528 Lys Phe Ser Thr Gly Gly Ser Lys Arg Pro Ala He Trp He Asp Thr 165 170 175 GGC ATC CAT TCC CGG GAG TGG GTC ACC CAG GCC AGT GGG GTC TGG ΗT 576 Gly He His Ser Arg Glu Trp Val Thr Gin Ala Ser Gly Val Trp Phe 180 185 190 GCA AAG AAG ATC ACT CAA GAC TAT GGG CAG GAT GCA GCT πC ACC GCC 624 Ala Lys Lys He Thr Gin Asp Tyr Gly Gin Asp Ala Ala Phe Thr Ala 195 200 205 Aπ CTC GAC ACC πG GAC ATC πC CTG GAG ATC GTC ACC AAC CCT GAT 672 He Leu Asp Thr Leu Asp He Phe Leu Glu He Val Thr Asn Pro Asp 210 215 220 GGC TTT GCC πC ACG CAC AGC ACG AAT CGC ATG TGG CGC AAG ACT CGG 720 Gly Phe Ala Phe Thr His Ser Thr Asn Arg Met Trp Arg Lys Thr Arg 225 230 235 240 TCC CAC ACA GCA GGC TCC CTC TGT Aπ GGC GTG GAC CCC AAC AGG AAC 768 Ser His Thr Ala Gly Ser Leu Cys He Gly Val Asp Pro Asn Arg Asn 245 250 255 TGG GAC GCT GGC HT GGG πG TCC GGA GCC AGC AGT AAC CCC TGC TCG 816 Trp Asp Ala Gly Phe Gly Leu Ser Gly Ala Ser Ser Asn Pro Cys Ser 260 265 270 GAG ACT TAC CAC GGC AAG Tπ GCC AAT TCC-GAA GTG GAG GTC AAG TCC 864 Glu Thr Tyr His Gly Lys Phe Ala Asn Ser Glu Val Glu Val Lys Ser 275 280 285 Aπ GTA GAC HT GTG AAG GAC CAT GGG AAC ATC AAG GCC πC ATC TCC 912 He Val Asp Phe Val Lys Asp His Gly Asn He Lys Ala Phe He Ser 290 295 300 ATC CAC AGC TAC TCC CAG CTC CTC ATG TAT CCC TAT GGC TAC AAA ACA 960 He His Ser Tyr Ser Gin Leu Leu Met Tyr Pro Tyr Gly Tyr Lys Thr 305 310 315 320 GAA CCA GTC CCT GAC CAG GAT GAG CTG GAT CAG Cπ TCC AAG GCT GCT 1008 Glu Pro Val Pro Asp Gin Asp Glu Leu Asp Gin Leu Ser Lys Ala Ala 325 330 335 GTG ACA GCC CTG GCC TCT CTC TAC GGG ACC AAG πC AAC TAT GGC AGC 1056 Val Thr Ala Leu Ala Ser Leu Tyr Gly Thr Lys Phe Asn Tyr Gly Ser 340 345 350 ATC ATC AAG GCA Aπ TAT CAA GCC AGT GGA AGC ACT Aπ GAC TGG ACC 1104 He He Lys Ala He Tyr Gin Ala Ser Gly Ser Thr He Asp Trp Thr 355 360 365 TAC AGC CAG GGC ATC AAG TAC TCC πC ACC πC GAG CTC CGG GAC ACT 1152 Tyr Ser Gin Gly He Lys Tyr Ser Phe Thr Phe Glu Leu Arg Asp Thr 370 375 380 GGG CGC TAT GGC πC CTG CTG CCA GCC TCC CAG ATC ATC CCC ACA GCC 1200 Gly Arg Tyr Gly Phe Leu Leu Pro Ala Ser Gin He He Pro Thr Ala 385 390 395 400 AAG GAG ACG TGG CTG GCG CTT CTG ACC ATC ATG GAG CAC ACC CTG AAT 1248 Lys Glu Thr Trp Leu Ala Leu Leu Thr He Met Glu His Thr Leu Asn 405 410 415 CAC CCC TAC 1257 His Pro Tyr (2) INFORMATION FOR SEQ ID NO: 2: (i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 419 amino acids (B) TYPE: amino acid (D) TOPOLOGY: l inear (i i ) MOLECULE πPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Arg Gly Leu Leu Val Leu Ser Val Leu Leu Gly Al a Val Phe Gly 1 5 10 15 Lys Gl u Asp Phe Val Gly His Gin Val Leu Arg He Ser Val Al a Asp 20 25 30 Gl u Al a Gin Val Gin Lys Val Lys Glu Leu Glu Asp Leu Glu His Leu 35 40 45 Gin Leu Asp Phe Trp Arg Gly Pro Ala His Pro Gly Ser Pro He Asp 50 55 60 Val Arg Met Pro Phe Pro Ser He Gin Al a Val Lys He Phe Leu Glu 65 70 75 80 Ser His Gly He Ser Tyr Glu Thr Met He Gl u Asp Val Gin Ser Leu 85 90 95 Leu Asp Glu Glu Gi n Glu Gin Met Phe Ala Phe Arg Ser Arg Al a Arg 100 105 110 Ser Thr Asp Thr Phe Asn Tyr Al a Thr Tyr His Thr Leu Glu Glu He 115 120 125 Tyr Asp Phe Leu Asp Leu Leu Val Ala Glu Asn Pro His Leu Val Ser 130 135 140 Lys He Gin He Gly Asn Thr Tyr Glu Gly Arg Pro He Tyr Val Leu 145 150 155 160 Lys Phe Ser Thr Gly Gly Ser Lys Arg Pro Al a He Trp He Asp Thr 165 170 175 Gly He His Ser Arg Glu Trp Val Thr Gin Ala Ser Gly Val Trp Phe 180 185 190 Al a Lys Lys He Thr Gin Asp Tyr Gly Gin Asp Ala Al a Phe Thr Ala 195 200 205 He Leu Asp Thr Leu Asp He Phe Leu Gl u He Val Thr Asn Pro Asp 210 215 220 Gly Phe Ala Phe Thr His Ser Thr Asn Arg Met Trp Arg Lys Thr Arg 225 230 235 240 Ser His Thr Al a Gly Ser Leu Cys He Gly Val Asp Pro Asn Arg Asn 245 250 255 Trp Asp Al a Gly Phe Gly Leu Ser Gly Al a Ser Ser Asn Pro Cys Ser 260 265 270 Glu Thr Tyr His Gly Lys Phe Ala Asn Ser Glu Val Glu Val Lys Ser 275 280 285 He Val Asp Phe Val Lys Asp His Gly Asn He Lys Al a Phe He Ser 290 295 300 He His Ser Tyr Ser Gin Leu Leu Met Tyr Pro Tyr Gly Tyr Lys Thr 305 310 315 320 Glu Pro Val Pro Asp Gin Asp Gl u Leu Asp Gin Leu Ser Lys Al a Ala 325 330 335 Val Thr Al a Leu Al a Ser Leu Tyr Gly Thr Lys Phe Asn Tyr Gly Ser 340 345 350 He He Lys Al a He Tyr Gin Al a Ser Gly Ser Thr He Asp Trp Thr 355 360 365 Tyr Ser Gin Gly He ' Lys Tyr Ser Phe Thr Phe Gl u Leu Arg Asp Thr 370 375 380 Gly Arg Tyr Gly Phe Leu Leu Pro Al a Ser Gi n He He Pro Thr Al a 385 390 395 400 Lys Glu Thr Trp Leu Al a Leu Leu Thr He Met Glu His Thr Leu Asn 405 410 415 His Pro Tyr (2) INFORMATION FOR SEQ ID NO: 3: (i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1251 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: l inear (ii) MOLECULE πPE: cDNA to mRNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1251 (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATG AGG πG ATC CTG Tπ Tπ GGT GCC CTT ITT GGG CAT ATC TAC TGT 48 Met Arg Leu He Leu Phe Phe Gly Ala Leu Phe Gly His He Tyr Cys 1 5 10 15 CTA GAA ACA Tπ GTG GGA GAC CAA Gπ Cπ GAG Aπ GTA CCA AGC AAT 96 Leu Glu Thr Phe Val Gly Asp Gin Val Leu Glu He Val Pro Ser Asn 20 25 30 GAA GAA CAA A AAA AAT CTG CTA CAA πG GAG GCT CAA GAA CAT CTC 144 Glu Glu Gin He Lys Asn Leu Leu Gin Leu Glu Ala Gin Glu His Leu 35 40 45 CAG Cπ GAT TTT TGG AAA TCA CCC ACC ACC TCA GGG GAG ACA GCC CAC 192 Gin Leu Asp Phe Trp Lys Ser Pro Thr Thr Ser Gly Glu Thr Ala His 50 55 60 GTC CGA Gπ CCC πC GTC AAC GTC CAG GCA GTC AAA GTG πC πG GAG 240 Val Arg Val Pro Phe Val Asn Val Gin Ala Val Lys Val Phe Leu Glu 65 70 75 80 TCC CAG GGA Aπ GCC TAT TCC ATC ATG Aπ GAA GAC GTG CAG GTC CTG 288 Ser Gin Gly He Ala Tyr Ser He Met He Glu Asp Val Gin Val Leu 85 90 95 πG GAC -AAA GAG AAT GAA GAA ATG ZTΪ Ul AAT AGG AGA AGA GAA CGG 336 Leu Asp Lys Glu Asn Glu Glu Met Leu Phe Asn Arg Arg Arg Glu Arg 100 105 110 AGT GGT AAC πC AAT TTT GGG GCC TAC CAT ACC CTG GAA GAG Aπ TCC 384 Ser Gly Asn Phe Asn ' Phe Gly Ala Tyr His Thr Leu Glu Glu He Ser 115 120 125 CAA GAA ATG GAT AAC CTC GTG GCT GAG CAC CCT GGT CTA GTG AGC AAA 432 Gin Glu Met Asp Asn Leu Val Ala Glu His Pro Gly Leu Val Ser Lys 130 135 140 GTG AAT Aπ GGC TCT TCT Tπ GAG AAC CGG CCT ATG AAC GTG CTC AAG 480 Val Asn He Gly Ser Ser Phe Glu Asn Arg Pro Met Asn Val Leu Lys 145 150 155 160 πC AGC ACC GGA GGA GAC AAG CCA GCT ATC TGG CTG GAT GCG GGG ATC 528 Phe Ser Thr Gly Gly Asp Lys Pro Ala He Trp Leu Asp Ala Gly He 165 170 175 CAT GCT CGA GAG TGG Gπ ACA CAA GCT ACG GCA CTT TGG ACA GCA AAT 576 His Ala Arg Glu Trp Val Thr Gin Ala Thr Ala Leu Trp Thr Ala Asn 180 185 190 AAG Aπ Gπ TCT GAT TAT GGA AAG GAC CCA TCC ATC ACT TCC Aπ CTG 624 Lys He Val Ser Asp Tyr Gly Lys Asp Pro Ser He Thr Ser He Leu 195 200 205 GAC GCC CTG GAT ATC πC CTC CTG CCA GTC ACA AAC CCT GAT GGA TAC 672 Asp Ala Leu Asp He Phe Leu Leu Pro Val Thr Asn Pro Asp Gly Tyr 210 215 220 GTG πC TCT CAA ACC AAA AAT CGT ATG TGG CGG AAG ACC CGG TCC AAG 720 Val Phe Ser Gin Thr Lys Asn Arg Met Trp Arg Lys Thr Arg Ser Lys 225 230 235 240 GTA TCT GGA AGC CTC TGT Gπ GGT GTG GAT CCT AAC CGG AAC TGG GAT 768 Val Ser Gly Ser Leu Cys Val Gly Val Asp Pro Asn Arg Asn Trp Asp 245 250 255 GCA GGT TTT GGA GGA CCT GGA GCC AGC AGC AAC CCT TGC TCT GAT TCA 816 Ala Gly Phe Gly Gly Pro Gly Ala Ser Ser Asn Pro Cys Ser Asp Ser 260 265 270 TAC CAC GGA CCC AGT GCC AAC TCT GAA Gπ GAA GTG AAA TCC ATA GTG 864 Tyr His Gly Pro Ser Ala Asn Ser Glu Val Glu Val Lys Ser He Val 275 280 285 GAC πC ATC AAG AGT CAT GGA AAA GTC AAG GCC πC Aπ ACC CTC CAC 912 Asp Phe He Lys Ser His Gly Lys Val Lys Ala Phe He Thr Leu His 290 295 300 AGC TAT TCC CAG CTG CTG ATG πC CCC TAT GGG TAC AAA TGT ACC AAG 960 Ser Tyr Ser Gin Leu Leu Met Phe Pro Tyr Gly Tyr Lys Cys Thr Lys 305 310 315 320 πA GAT GAC Tπ GAT GAG CTG AGT GAA GTG"GCC CAA AAG GCT GCC CAA 1008 Leu Asp Asp Phe Asp Glu Leu Ser Glu Val Ala Gin Lys Ala Ala Gin 325 330 335 TCT CTG AGA AGC CTG CAT GGC ACC AAG TAC AAA GTG GGA CCA ATC TGC 1056 Ser Leu Arg Ser Leu His Gly Thr Lys Tyr Lys Val Gly Pro He Cys 340 345 350 TCT GTC ATC TAC CAA GCC AGT GGA GGA AGC Aπ GAC TGG TCC TAT GAT 1104 Ser Val He Tyr Gin Ala Ser Gly Gly Ser He Asp Trp Ser Tyr Asp 355 360 365 TAT GGC ATC AAG TAC TCA Tπ GCC TTT GAA CTG AGA GAC ACA GGG CGC 1152 Tyr Gly He Lys Tyr Ser Phe Ala Phe Glu Leu Arg Asp Thr Gly Arg 370 375 380 TAC GGC πC CTC πG CCA GCC CGT CAG ATC CTG CCC ACA GCC GAG GAG 1200 Tyr Gly Phe Leu Leu Pro Ala Arg Gin He Leu Pro Thr Ala Glu Glu 385 390 395 400 ACC TGG Cπ GGC πG AAG GCA ATC ATG GAG CAT GTG CGA GAC CAC CCC 1248 Thr Trp Leu Gly Leu Lys Ala He Met Glu His Val Arg Asp His Pro 405 410 415 TAT 1251 Tyr (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 417 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE πPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Arg Leu He Leu Phe Phe Gly Ala Leu Phe Gly His He Tyr Cys 1 5 10 15 Leu Glu Thr Phe Val Gly Asp Gin Val Leu Glu He Val Pro Ser Asn 20 25 30 Glu Glu Gin He Lys Asn Leu Leu Gin Leu Glu Ala Gin Glu His Leu 35 40 45 Gin Leu Asp Phe Trp Lys Ser Pro Thr Thr Ser Gly Glu Thr Ala His 50 55 60 Val Arg Val Pro Phe Val Asn Val Gin Ala Val Lys Val Phe Leu Glu 65 70 75 80 Ser Gin Gly He Ala Tyr Ser He Met He Glu Asp Val Gin Val Leu 85 90 95 Leu Asp Lys Glu Asn Glu Glu Met Leu Phe Asn Arg Arg Arg Glu Arg 100 105 110 Ser Gly Asn Phe Asn Phe Gly Ala Tyr His Thr Leu Glu Glu He Ser 115 120 125 Gin Glu Met Asp Asn Leu Val Ala Glu His Pro Gly Leu Val Ser Lys 130 135 140 Val Asn He Gly Ser Ser Phe Glu Asn Arg Pro Met Asn Val Leu Lys 145 150 155 160 Phe Ser Thr Gly Gly Asp Lys Pro Ala He Trp Leu Asp Ala Gly He 165 170 175 His Ala Arg Glu Trp Val Thr Gin Ala Thr Ala Leu Trp Thr Ala Asn 180 185 190 Lys He Val Ser Asp Tyr Gly Lys Asp Pro Ser He Thr Ser He Leu 195 200 205 Asp Ala Leu Asp He Phe Leu Leu Pro Val Thr Asn Pro Asp Gly Tyr 210 215 220 Val Phe Ser Gin Thr Lys Asn Arg Met Trp Arg Lys Thr Arg Ser Lys 225 230 235 240 Val Ser Gly Ser Leu Cys Val Gly Val Asp Pro Asn Arg Asn Trp Asp 245 250 255 Ala Gly Phe Gly Gly Pro Gly Ala Ser Ser Asn Pro Cys Ser Asp Ser 260 265 270 Tyr His Gly Pro Ser Ala Asn Ser Glu Val Glu Val Lys Ser He Val 275 280 285 Asp Phe He Lys Ser His Gly Lys Val Lys Ala Phe He Thr Leu His 290 295 300 Ser Tyr Ser Gi n Leu Leu Met Phe Pro Tyr Gly Tyr Lys Cys Thr Lys 305 310 315 320 Leu Asp Asp Phe Asp Glu Leu Ser Glu Val Ala Gin Lys Ala Ala Gin 325 330 335 Ser Leu Arg Ser Leu His Gly Thr Lys Tyr Lys Val Gly Pro He Cys 340 345 350 Ser Val He Tyr Gin Ala Ser Gly Gly Ser He Asp Trp Ser Tyr Asp 355 360 365 Tyr Gly He Lys Tyr Ser Phe Al a Phe Gl u Leu Arg Asp Thr Gly Arg 370 375 380 Tyr Gly Phe Leu Leu Pro Al a Arg Gin He Leu Pro Thr Al a Gl u Glu 385 390 395 400 Thr Trp Leu Gly Leu Lys Al a He Met Glu His Val Arg Asp His Pro 405 410 415 Tyr (2) INFORMATION FOR SEQ ID NO: 5 : (i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii ) MOLECULE πPE: cDNA to mRNA (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CCTGπAπG GCTGCCCTAC π 22 (2) INFORMATION FOR SEQ ID NO: 6: (i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULE πPE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AAGCCAGGTC TCTTCTGCTG TC 22 22 (2) INFORMATION FOR SEQ ID NO: f: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE πPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GGTCCAGTCA GCAGTGCTTC C 21 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE πPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GAGCTCGAAG GCGAAGGAGT A 21 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE πPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GAGCTCGAAG CCGAAGGAGT A 21 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE πPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TCCCCCGGGC TGCAGGATAT CGATAGCTGG GCTGTGT 37 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (i i ) MOLECULE πPE: DNA (genomic) (xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 11 : ATCAAGTACT CCTCCCAGCT CCGGGAC 27 (2) INFORMATION FOR SEQ ID NO: 12: (i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (i i ) MOLECULE πPE: DNA (genomic) (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 12: πTATCAAGC CAGTGGAGGT AπGACTGGA CC 32 (2) INFORMATION FOR SEQ ID NO: 13: (i ) SEQUENCE CHARACTERISTICS : (A) LENGTH: 33 base pairs (B) TYPE : nuclei c acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (i i ) MOLECULE πPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: AAGGCAAITT ATCAAGGCAG TGGAAGCACT Aπ 33 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TCCCCCGGGC TGCAGGATAT CGATAGCTGG GCTGTGT 37 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE πPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GCTGAAGCπ CGGAGGACπ T 21 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE πPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TCπGACCGC CTGGATGCTG 20 (2) INFORMATION FOR SEQ ID NO: 17: (i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 419 amino acids (B) TYPE: amino acid (D) TOPOLOGY: l inear (ii ) MOLECULE πPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Met Arg Gly Leu Leu Val Leu Ser Val Leu Leu Gly Al a Val Phe Gly 1 5 10 15 Lys Gl u Asp Phe Val Gly His Gi n Val Leu Arg He Ser Val Al a Asp 20 25 30 Gl u Ala Gi n Val Gi n Lys Val Lys Gl u Leu Glu Asp Leu Gl u His Leu 35 40 45 Gin Leu Asp Phe Trp Arg Gly Pro Al a His Pro Gly Ser Pro He Asp 50 55 60 Val Arg Met Pro Phe Pro Ser He Gin Al a Val Lys He Phe Leu Glu 65 70 75 80 Ser His Gly He Ser Tyr Glu Thr Met He Gl u Asp Val Gi n Ser Leu 85 90 95 Leu Asp Glu Gl u Gin Glu Gin Met Phe Ala Phe Arg Ser Arg Al a Arg 100 105 110 Ser Thr Asp Thr Phe Asn Tyr Ala Thr Tyr His Thr Leu Glu Glu He 115 120 125 Tyr Asp Phe Leu Asp Leu Leu Val Ala Glu Asn Pro His Leu Val Ser 130 135 140 Lys He Gin He Gly Asn Thr Tyr Glu Gly Arg Pro He Tyr Val Leu 145 150 155 160 Lys Phe Ser Thr Gly Gly Ser Lys Arg Pro Ala He Trp He Asp Thr 165 170 175 Gly He His Ser Arg Glu Trp Val Thr Gin Al a Ser Gly Val Trp Phe 180 185 190 Ala Lys Lys He Thr Gin Asp Tyr Gly Gin Asp Al a Al a Phe Thr Ala 195 200 205 He Leu Asp Thr Leu Asp He Phe Leu Gl u He Val Thr Asn Pro Asp 210 215 220 Gly Phe Al a Phe Thr His Ser Thr Asn Arg Met Trp Arg Lys Thr Arg 225 230 235 240 Ser His Thr Al a Gly Ser Leu Cys He Gly Val Asp Pro Asn Arg Asn 245 250 255 Trp Asp Al a Gly Phe Gly Leu Ser Gly Ala Ser Ser Asn Pro Cys Ser 260 265 270 Gl u Thr Tyr His Gly Lys Phe Al a Asn Ser Gl u Val Gl u Val Lys Ser 275 280 285 He Val Asp Phe Val Lys Asp His Gly Asn He Lys Al a Phe He Ser 290 295 300 He His Ser Tyr Ser Gin Leu Leu Met Tyr Pro Tyr Gly Tyr Lys Thr 305 310 315 320 Glu Pro Val Pro Asp Gin Asp Glu Leu Asp Gin Leu Ser Lys Al a Ala 325 330 335 Val Thr Al a Leu Al a Ser Leu Tyr Gly Thr Lys Phe Asn Tyr Gly Ser 340 345 350 He He Lys Al a He Tyr Gin Ala Ser Gly Ser Thr He Asp Trp Thr 355 360 365 Tyr Ser Gin Gly He lys Tyr Ser Phe Gly Phe Glu Leu Arg Asp Thr 370 375 380 Gly Arg Tyr Gly Phe Leu Leu Pro Ala Ser Gin He He Pro Thr Ala 385 390 395 400 Lys Glu Thr Trp Leu Ala Leu Leu Thr He Met Gl u His Thr Leu Asn 405 410 415 His Pro Tyr (2) INFORMATION FOR SEQ ID NO: 18: (i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 417 amino acids (B) TYPE: amino acid (D) TOPOLOGY: l inear (ii ) MOLECULE πPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Met Arg Leu He Leu Phe Phe Gly Ala Leu Phe Gly His He Tyr Cys 1 5 10 15 Leu Gl u Thr Phe Val Gly Asp Gin Val Leu Glu He Val Pro Ser Asn 20 25 30 Glu Glu Gin He Lys Asn Leu Leu Gin Leu Glu Ala Gin Glu His Leu 35 40 45 Gin Leu Asp Phe Trp Lys Ser Pro Thr Thr Ser Gly Glu Thr Al a His 50 55 60 Val Arg Val Pro Phe Val Asn Val Gin Ala Val Lys Val Phe Leu Glu 65 70 ' 75 80 Ser Gin Gly He Al a Tyr Ser He Met He Gl u Asp Val Gi n Val Leu 85 90 95 Leu Asp Lys Gl u Asn Gl u Gl u Met Leu Phe Asn Arg Arg Arg Gl u Arg 100 105 110 Ser Gly Asn Phe Asn Phe Gly Al a Tyr His Thr Leu Glu Gl u He Ser 115 120 125 Gi n Gl u Met Asp Asn Leu Val Al a Glu His Pro Gly Leu Val Ser Lys 130 135 140 Val Asn He Gly Ser Ser Phe Gl u Asn Arg Pro Met Asn Val Leu Lys 145 150 155 160 Phe Ser Thr Gly Gly Asp Lys Pro Ala He Trp Leu Asp Al a Gly He 165 170 175 His Al a Arg Gl u Trp Val Thr Gin Ala Thr Al a Leu Trp Thr Al a Asn 180 185 190 Lys He Val Ser Asp Tyr Gly Lys Asp Pro Ser He Thr Ser He Leu 195 200 205 Asp Al a Leu Asp He Phe Leu Leu Pro Val Thr Asn Pro Asp Gly Tyr 210 215 220 Val Phe Ser Gin Thr Lys Asn Arg Met Trp Arg Lys Thr Arg Ser Lys 225 230 235 240 Val Ser Gly Ser Leu Cys Val Gly Val Asp Pro Asn Arg Asn Trp Asp 245 250 255 Al a Gly Phe Gly Gly Pro Gly Al a Ser Ser Asn Pro Cys Ser Asp Ser 260 265 270 Tyr His Gly Pro Ser Ala Asn Ser Glu Val Glu Val Lys Ser He Val 275 280 285 Asp Phe He Lys Ser His Gly Lys Val Lys Ala Phe He Thr Leu His 290 295 300 Ser Tyr Ser Gin Leu Leu Met Phe Pro Tyr Gly Tyr Lys Cys Thr Lys 305 310 315 320 Leu Asp Asp Phe Asp Gl u Leu Ser Glu Val Al a Gin Lys Al a Al a Gin 325 330 335 Ser Leu Arg Ser Leu His Gly Thr Lys Tyr Lys Val Gly Pro He Cys 340 345 350 Ser Val He Tyr Gin Al a Ser Gly Gly Ser He Asp Trp Ser Tyr Asp 355 360 365 Tyr Gly He Lys Tyr Ser Phe Gly Phe Glu Leu Arg Asp Thr Gly Arg 370 375 380 Tyr Gly Phe Leu Leu Pro Al a Arg Gin He Leu Pro Thr Al a Glu Glu 385 390 395 400 Thr Trp Leu Gly Leu Lys Ala He Met Glu His Val Arg Asp His Pro 405 410 415 Tyr (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE πPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GCAGAAGCπ CAGAAACGπ TGTGGGAGAT CAAGTTCTTG AGAπGTACC 50 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE πPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: CTCTπGTCC AACAGGACCT G 21 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii ) MOLECULE πPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TCCTCCACTG CCTTGGTAGA T 21 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (i i ) MOLECULE πPE: DNA (genomic) (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 22 : CAGπCAAAG CCAAATGAGT A 21

Claims: CLAIMS 1. A conjugate comprising a targetting molecule capable of delivering an enzyme to a particular mammalian cell and a mammalian enzyme conjugated to the targetting molecule and capable of catalysing a functionally inactive precursor of a chemotherapeutic agent to its active form wherein the enzyme is a mutant and the precursor is refractory to endogenous catalysis by the wild-type form of the enzyme. 2. A conjugate according to Claim 1, wherein the enzyme is one of a hydrolase, a transferase, an oxido-reductase, an isomerase, a lyase and a ligase. 3. A conjugate according to Claim 2, wherein the hydrolase is a peptidase. 4. A conjugate according to Claim 2, wherein the hydrolase is an esterase. 5. A conjugate according to Claim 3, wherein the peptidase is an exopeptidase. 6. A conjugate according either Claim 3 or 4, wherein the peptidase of Claim 3 or the esterase of Claim 4 is a carboxypeptidase. 7. A conjugate according to Claim 6 wherein the carboxypeptidase is a human carboxypeptidase A. 8. A conjugate according to Claim 7 wherein the human carboxypeptidase A is HCPA 1 or HCPA 2 possessing amino acid substitutions at one or more of residues 203, 210, 242, 244, 250, 253, 255, 267, 268, 269 and 305. 9. A conjugate according to Claim 8 wherein the amino acid substitutions are selected from the following: Gly at 250 and 268, Gly at 253 and 268, Gly at 250 and His at 268, Gly at 250, Ala at 255 and His 268, His at 268, and Gly at 268. 10. A conjugate according to Claim 9 wherein the substitution is Gly at 268. 11. A conjugate according to any of Claims 8 to 10 wherein all other residues are wild-type. 12. A conjugate according to any of Claims 1 to 11 wherein the targetting molecule is one of an antibody, a hormone, a ligand, a cytokine, an antigen, an oligonucleotide and a peptidomimetic. 13. A conjugate according to Claim 12 wherein the antibody is a polyclonal antibody, a monoclonal antibody or a fragment thereof. 14. A conjugate according to Claim 13 wherein the antibody or fragment thereof is humanised. 15. A conjugate according to any of Claims 1 to 14, wherein the targetting molecule is joined to the enzyme by a linker molecule. 16. A conjugate according to any of Claims 1 to 14 which is translated from a single RNA transcript or multiple RNA transcripts. 17. A DNA or RNA molecule encoding a conjugate as claimed in Claim 16. 18. A vector containing the DNA or RNA claimed in Claim 17. 19. A cell line containing the DNA or RNA claimed in Claim 17 or a vector as claimed in Claim 18. 20. A compound of general formula II F B-D-E-F-X - C — CO 2 H (π) I R 2a wherein, X represents NH or O and R 2 represents CO 2 H, SO3H, SO 2 H, OSO3H, PO 3 H 2 , OPO 3 H 2 , C . 6 alkyl esterified CO2H, Cι_g esterified PO3H2, C4..12 alkyl C4.12 alkenyl, C4.12 alkynyl, C4..12 branched alkyl; C g alkyl or C3_g branched alkyl; substituted with CO 2 H, SO3H, SO 2 H, OSO3H, PO 3 H 2 , OPO 3 H 2 , C β alkyl esterified CO2H, Cι_6 alkyl esterified PO3H2, hydroxyl, alkoxy, halo, haloalkyl, cyano or carboxamide; C3_ cycloalkyl, aryl or heteroaryl optionally substituted with C\ _8 alkyl, C3_ branched alkyl, C3_g cycloalkyl, C3.6 alkenyl, C2-6 alkynyl, C4.6 cycloalkenyl, trisubstituted-silyl i.e. (R 13 )(R 1 )(R 15 )Si, where R 13 , R 14 , and R 1 - 5 are Cι_6 alkyl, C_.._, branched alkyl, C3.6 cycloalkyl, C3.6 alkenyl, C2-6 alkynyl, C4.6 cycloalkenyl, aryl, or heteroaryl, and where R* 3 , R 4 , and R^- are each the same group or different groups (e.g. trimethylsilyl, tert- butyldimethylsilyl, cyclohexyldimethylsilyl, or phenyldimethylsilyl), aryl, CO2H, SO3H, SO 2 H, OSO3H, PO 3 H 2 , OPO 3 H 2 , Cι_ 6 alkyl esterified CO 2 H, C^ alkyl esterified PO3H2, carboxamide, hydroxyl, C\. alkoxy, C\. alkythio, mercapto, halo, haloalkyl, nitro or cyano, R 2a and R 2 ° represent H, hydroxy, mercapto, alkoxy, alkylthio, halo, cyano, CO2H, SO3H, SO2H, OSO3H, PO3H2, OPO3H2, C]_6 alkyl esterified CO2H, carboxamide, C\.β alkyl esterified PO3H2, cyclic C2-6 [i* e ** R^ a and R > together represent (CH2)2-6_I* trisubstituted-silyl i.e. (R 13 )(R 14 )(R 15 )Si, where R 13 , R 14 , and R 15 are C\. alkyl, 03.5 branched alkyl, C3.6 cycloalkyl C3.6 alkenyl; C2-6 alkynyl, C4.6 cycloalkenyl, aryl, or heteroaryl and where R^ 3 , R 4 , and R ~ could be the same group or different groups, e.g. trimethylsilyl, tert-butyldimethylsilyl, cyclohexyldimethylsilyl, or phenyldimethylsilyl; or C ^ alkyl or C]_6 branched alkyl or Cj_6 cycloalkyl or aryl or heteroaryl optionally substituted with hydroxy, mercapto, Cι_6 alkoxy, Cι_6 alkylthio, halo, cyano, CO2H, SO3H, SO2H, OSO3H nitro, PO3H2, OPO3H2, Cι_6 alkyl esterified CO2H, carboxamide, Cι_6 alkyl esterified PO3H2, Cι_6 alkyl, C g branched alkyl, C\. 6 cycloalkyl, trisubstituted-silyl i.e.(R 13 )(R 14 )(R 15 )Si, where R 13 , R 14 , and Rl5 are Cj_6 alkyl, C3.6 branched alkyl, C3.6 cycloalkyl, aryl, or heteroaryl, and where R^ 3 , R 4 , and )~ could be the same group or different groups, e.g. trimethylsilyl, tertbutyldimethylsilyl or phenyldimethylsilyl; with the proviso that R2a and R 2D are not both hydroxy or mercapto. F comprises a moiety of formula (IN) wherein Rl represents a group corresponding to the side chain of any α- amino acid for example, H, Cι.β alkyl, Cι.g alkenyl, C g alkynyl optionally substituted with CO 2 H, Cι_6 alkyl esterified CO2H, OPO3, PO3H2, Cι_6 alkyl esterified PO3H2, halo, hydroxy, carboxamide, amino optionally substituted with Cι_6 alkyl, cyano or nitro, E represents where n = 3-6 or each of which may be optionally substituted with one or more of hydroxy, one or more of alkoxyl, halo, or C \. alkyl optionally substituted with one or more of hydroxy, Cι_6 alkoxy or halo. D represents -CH 2 -CH(R 3 )-, -CH 2 -NR 3 -, -NR 3 -CH 2 -, -CH 2 S- or -CH 2 O- where R 3 is H, Cj^alkyl, allyl or propargyl, optionally substituted with one or more of Cι_g alkoxy halo, hydroxy or cyano; and B represents wherein, R 4 represents H, C3.6 branched alkyl, C3.6 cycloalkyl, or C\. alkyl; R represents Cj_6 alkyl, C3.6 branched alkyl, C3.6 cycloalkyl, amino (optionally substituted with Cι_6 alkyl, C\. alkanoyl or benzyl); R > represents H, Cι_6 alkyl, C^- branched alkyl, C3.6 cycloalkyl, amino (optionally substituted with Cι_g alkyl, Cj.6 alkanoyl or benzyl), Cι_6 alkoxyl or Cι_6 alkylthio; and R^ represents H, \. alkyl, 03.5 branched alkyl, C3_6 cycloalkyl, halo C\._ alkyl or halo and salts N-oxides, solvates and physiologically functional derivatives thereof; with the proviso that the following compounds are not included within the formula. N-(N*^4-(((2-_m_ino-3,4-dihydro-4-oxo-6-quinazolinyl)methyl)(2- propynyl)-imino)benzoyl)-L-glutam- 1 -yl)-L-glut__π_ic acid, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-L- glutamic acid, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl- L-aspartic acid, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -y 1 - L-phenylalanine, 4-bis(2-chloroethyl)amino)-phenylalanylphenylalanine and 4-bis(2-chloroethyl)amino)-phenylalanyl-3,5-dimethyl-4-methoxyphenylalanine 21. A compound according to Claim 20 and salts, N-oxides, solvates and physiologically functional derivatives thereof wherein X is H; R 2 is C3_ cycloalkyl or aryl substituted with Ci.galkyl, C3.gcycl0al.cyl, C3.9 branched alkyl or trisubstituted-silyl i.e. (R 13 )(R 1 )(R 15 )Si, where R 13 , R 14 , and R 15 are Cj.6 alkyl, C3.6 branched alkyl, C3.6 cycloalkyl, aryl, or heteroaryl, and where R*^ 3 , R** 4 , and R^ are each the same group or different groups (e.g. trimethylsilyl, tert-butyldimethylsilyl, cyclohexyldimethylsilyl, or phenyldimethylsilyl); R 2a and R 2b are both H; R 1 is H, C1.6 alkyl, CH 2 CO 2 H, CH 2 CH 2 CO 2 H or CH2,CH 2 CH 2 NH2, E is D is -CH 2 NH-, -CH 2 N(CH 3 )-, -CH 2 N(CH 2 C≡CH)-, -CH 2 CH 2 - or -CH 2 -CH(C 2 H 5 )- and B is 22. A compound according to Claim 20 and salts, N-oxides, solvates and physiologically functional derivatives thereof wherein X is NH; R 2 is phenyl or para-hydroxyphenyl substituted with cyclopentyl, cyclobutyl or tert-butyl. The cyclopentyl, cyclobutyl and tert-butyl groups may both be either ortho- or meta- but are preferably meta-, R 2a and R 2b are H, R 1 is CH 2 CH2CO 2 H D is -CH 2 N (CH 3 ) . and B is 23. The following compounds and salts, N-oxides, solvates and physiologically functional derivatives thereof: N-((S)~4-carboxy-2-(5-((( 1 ,2-dihydro-3-methyl- 1 -oxobenzo(F)quinazolin-9- yl)methyl)amino)- 1 -oxo-2-isoindolinyl)butanoyl)-L-phenylalanine, N-(4-(((2,4-di__mmo-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-2- cyclopentyl-L-phenylalanine, N*^4-(((2,4-di__mino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl- 3-cyclopentyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl- 3 -cyclobutyl-L-phenylalanine, N*^4-(((2,4-dian_dno-6-pteridinyl)methyl)me ylamino)benzoyl)-L-glutam- 1 -yl- 3-trimethylsilyl-L-phenylalanine, N*^4-(((2,4-dian_ no-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3- tert-butyl-L-phenylalanine, N*^4-((3-(2,4-diammo-l,6^dihydro-6^oxo-5-pyrimidinyl)propyl)an_ino)benzoyl)- L-glutam- 1 -yl-3 -cyclopentyl-L-phenylalanine, N-(4-((( 1 ,2-dihydro-3-methyl- 1 -oxobenzo(F)quinazolin-9-yl)methyl)amino)-2- fluorobenzoyl)-L-glutam- 1 -yl-3 -cyclopentyl-L-phenylalanine, N-(4-(((2,4-diamino-6-pteridinyl-methyl)methylamino)benzoyl)-L-glutam-l-yl-3- cyclopentyl-L-tyrosine, N*^4-(((2,4-di_unino-6-pteridinyl)methyl)methyl_ιmino)benzoyl)-L-glutam-l-yl- 3,5-diiodo-L-tyrosine, N-(4-(((2,4-diaπ___no-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-2- cyclopentyl-L-tyrosine, (S)-3-(3-cyclopentyl-4-hydroxyphenyl)-2-((N-(4-(((2,4-diamino-6- pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l -yl)oxy)propionic acid, N-(4*^((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam-l-yl-3- tert-butyl-L-tyrosine. 24. N-(4-(((2,4-di_ιmino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutam- 1 -yl-3- cylopentyl-L-tyrosine and salts, N-oxides, solvates and physiologically functional derivatives thereof. 25. The use of a compound according to any of Claims 20 to 24 or of a conjugate according to any of Claims 1 to 16 in medical therapy. 26. The use of a compound according to any of Claims 20 to 24 or of a conjugate according to any of Claims 1 to 16 or of a cell line according to Claim 19 or of a vector according to Claim 18 or of DNA or RNA according to Claim 17 in the preparation of a medicament for use in medical therapy. 27. A method of targetted chemotherapy consisting of the administration of a conjugate comprising a mutant mammalian enzyme and a cell-type specific targetting molecule wherein the enzyme is capable of catalysing a functionally inactive precursor of a chemotherapeutic agent to its active form which said precursor is refractory to endogenous catalysis. 28. The use of a precursor of a chemotherapeutic agent in the preparation of a medicament for use in the treatment of a patient requiring therapy to a particular cell population who has a conjugate as claimed in any of Claims 1 to 16 bound to that particular cell-population. 29. A mutant mammalian enzyme capable of catalysing a functionally inactive precursor of a chemotherapeutic agent to its active form wherein the precursor is refractory to endogenous catalysis. 30. An enzyme according to Claim 29 wherein the enzyme possesses carboxypeptidase activity. 31. An enzyme according to Claim 30 wherein the enzyme is a human carboxypeptidase A. 32. An enzyme according to Claim 31 wherein the enzyme is HCPA 1 or HCPA 2 possessing amino acid substitutions at one or more of residues 203, 210, 242, 244, 250, 253, 255, 267, 268, 269 and 305. 33. An enzyme according to Claim 32 wherein the amino acid substitutions are selected from the following: Gly at 250 and 268, Gly at 253 and 268, Gly at 250 and His at 268, Gly at 250, Ala at 255 and His 268, His at 268, and Gly at 268. 34. An enzyme according to Claim 33 wherein the substitution is Gly at 268. 35. An enzyme according to any of Claims 32 to 34 wherein all other residues are wild-type. 36. A DNA or RNA molecule encoding an enzyme claimed in any of Claims 29 to 35. 37. A vector containing a DNA or RNA molecule claimed in Claim 36. 38. A cell line combining a DNA or RNA molecule as claimed in Claim 36 or a vector as claimed in Claim 37.

Inventors: BLUMENKOPF TODD ANDREW, CORY MICHAEL, SMITH GARY KEITH, BLUMENKOPF, TODD, ANDREW, CORY, MICHAEL, SMITH, GARY, KEITH
Applicants: BLUMENKOPF TODD ANDREW, CORY MICHAEL, SMITH GARY KEITH, WELLCOME FOUND, BLUMENKOPF, TODD, ANDREW, CORY, MICHAEL, SMITH, GARY, KEITH, THE WELLCOME FOUNDATION LIMITED
Application year: 1994
Main classification: A61K47
IPC codes: A61K38, A61K47, C12N1, C12N9, C12N15, C12R1